Anti-PD-L1 antibody for detecting PD-L1

ABSTRACT

The present invention provides an anti-PD-L1 antibody capable of staining tumor cells such as melanoma cells. 
     An anti-PD-L1 antibody comprising (a) a light chain comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5). A composition for detecting PD-L1, comprising the above antibody as an active ingredient. A method for preparing the above antibody is also provided.

PRIORITY APPLICATIONS

This application is a U.S. National Stage Filing under 35 U.S.C. 371 from International Application No. PCT/JP2018/011895, filed on Mar. 23, 2018, and published as WO2018/181064 on Oct. 4, 2018, which claims the benefit of priority to Japanese Application No. 2017-061389, filed on Mar. 27, 2017; the benefit of priority of each of which is hereby claimed herein, and which applications and publication are hereby incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to an anti-PD-L1 antibody for detecting PD-L1.

BACKGROUND ART

Malignant melanoma originating from melanocytes is one of the most commonly observed malignant tumors in the canine oral cavity (Non-Patent Document No. 1: Todoroff et al., J Am Vet Med Assoc. 1979 Sep. 15: 175(6):567-71). Since this type of melanoma generally tends to be highly invasive and metastatic, early diagnosis and treatment are desired. On the other hand, malignant melanoma has a wide tissue variation, presenting various morphologies such as epithelial-like, round cell-like or fibrosarcoma-like morphology. Thus, malignant melanoma is one of those tumors which involve difficulty in tissue diagnosis. Although confirmation of melanin pigment is important for their diagnosis, a large number of malignant melanomas do not have melanin pigment and, sometimes, diagnosis cannot be made with histological observations alone. This has led to searches for diagnostic markers that can be used in immunohistochemical techniques. Among such markers, Melan A/MART-1, vimentin, S100, neuron-specific enolase and the like have been reported to be useful (Non-Patent Document No. 2: Ramos-Vara et al., Vet Pathol. 2000 November; 37(6):597-608). However, even Melan A/MART-1, the most widely used diagnostic marker, has a positive rate not higher than about 60% which varies among reports (Non-Patent Document No. 3: Koenig et al., Vet Pathol. 2001 July; 38(4):427-35). Because of this sensitivity problem, the utility of Melan A/MART-1 in actual diagnosis is still arguable. Further. Melan A/MART-1 is not stained in amelanotic melanoma (Non-Patent Document No. 3: Koenig et al., Vet Pathol. 2001 July; 38(4):427-35), so its application to diagnosis is limited. Under these circumstances, it is desired to develop highly sensitive, novel diagnostic markers to malignant melanoma.

PRIOR ART LITERATURE Non-Patent Documents

Non-Patent Document No. 1: Todoroff et al., J Am Vet Med Assoc. 1979 Sep. 15; 175(6):567-71

Non-Patent Document No. 2: Ramos-Vara et al., Vet Pathol. 2000 November; 37(6):597-608

Non-Patent Document No. 3: Koenig et al., Vet Pathol. 2001 July; 38(4):427-35

DISCLOSURE OF THE INVENTION Problem for Solution by the Invention

It is an object of the present invention to provide a PD-L1 antibody capable of staining tumor cells such as melanoma cells.

Means to Solve the Problem

The present inventors have established a number of monoclonal antibodies which react with the PD-L1 protein of various animals. It has been revealed that, among those monoclonal antibodies, a rat anti-bovine PD-L1 monoclonal antibody (6C11-3A11) is capable of staining melanoma tumor cells very strongly. Currently, this monoclonal antibody is used for selecting candidate dogs for therapy with chimeric antibodies. The subject PD-L1 antibody (6C11-3A11) is also capable of immunohistochemically staining ovine, porcine and bovine PD-L1 proteins. Further, the present inventors have determined the CDRs (complementarity-determining regions) of the variable regions of the subject PD-L1 antibody (6C11-3A11). The present invention has been achieved based on these findings.

A summary of the present invention is as described below.

-   (1) An anti-PD-L1 antibody comprising (a) a light chain comprising     CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2     having the amino acid sequence of SGS and CDR3 having the amino acid     sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain     comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID     NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID     NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF     (SEQ ID NO: 5). -   (2) The antibody of (1) above, which is derived from rat. -   (3) The antibody of (2) above, which is a rat anti-bovine PD-L1     antibody. -   (4) The antibody of (3) above, wherein the light chain variable     region has the amino acid sequence as show in SEQ ID NO. 6 and the     heavy chain variable region has the amino acid sequence as shown in     SEQ ID NO: 7. -   (5) The antibody of any one of (1) to (4) above, wherein the light     chain constant region has the amino acid sequence of the constant     region of kappa chain. -   (6) The antibody of any one of (1) to (5) above, wherein the heavy     chain constant region has the amino acid sequence of the constant     region of IgG2a. -   (7) The antibody of (5) or (6) above, wherein the light chain     constant region has the amino acid sequence as shown in any one of     SEQ ID NOS: 8, 10 to 12 and the heavy chain constant region has the     amino acid sequence as shown in SEQ ID NO: 9 or 13. -   (8) The antibody of anyone of (1) to (7) above which has a     four-chain structure comprising two light chains and two heavy     chains. -   (9) A composition for detecting PD-L1, comprising the antibody of     any one of (1) to (8) above as an active ingredient. -   (10) The composition of (9) above for use in diagnosis of cancers     and/or inflammations. -   (11) The composition of (10) above, wherein the cancers and/or     inflammations are selected from the group consisting of neoplastic     diseases, leukemia, Johne's disease, anaplasmosis, bacterial     mastitis, mycotic mastitis, mycoplasma infections (such as     mycoplasma mastitis, mycoplasma pneumonia or the like),     tuberculosis, Theileria orientalis infection, cryptosporidiosis,     coccidiosis, trypanosomiasis and leishmaniasis. -   (12) The composition of (9) above for use in selecting subject     animals suitable for therapy with anti-PD-L1 antibodies. -   (13) A DNA encoding the anti-PD-L1 antibody of (1) above. -   (14) A vector comprising the DNA of (13) above. -   (15) A host cell transformed with the vector of (14) above. -   (16) A method of preparing an antibody, comprising culturing the     host cell of (15) above and collecting an anti-PD-L1 antibody from     the resultant culture. -   (17) A DNA encoding the light chain of an anti-PD-L1 antibody, said     light chain comprising CDR1 having the amino acid sequence of KSISKY     (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3     having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2). -   (18) A DNA encoding the heavy chain of an anti-PD-L1 antibody, said     heavy chain comprising CDR1 having the amino acid sequence of     GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of     INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of     ARGTMMVVISHWKFDF (SEQ ID NO: 5).

Effect of the Invention

According to the present invention, a novel anti-PD-L1 antibody capable of staining tumor cells, such as melanoma cells, has been obtained.

The present specification encompasses the contents disclosed in the specification and/or the drawings of Japanese Patent Application No. 2017-61389 based on which the present patent application claims priority.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

FIG. 1 Binding specificity of rat anti-bovine PD-L1 antibody 6C11-3A11. Rat anti-bovine PD-L1 antibody 6C11-3A11 did not bind to EGFP expressing cells, but specifically bound to canine PD-L1-EGFP expressing cells.

FIG. 2 Predicted CDR regions of rat anti-bovine PD-L1 antibody 6C11-3A11. The regions of CDR1. CDR2 and CDR3 in the light chain variable region and the heavy chain variable region of rat anti-bovine PD-L1 antibody 6C1-3A11 are shown.

FIG. 3 Comparative immunohistochemical staining images of canine melanoma. Left: stained with a commercial antibody (MelanA antibody). Tumor cells were stained very weakly. Right: stained with the PD-L1 antibody 6C11-3A11 established by the present inventors. Tumor cells were stained very strongly.

FIG. 4 Immunohistochemical staining image of canine melanoma.

FIG. 5-1 Immunohistochemical staining images of other tumors. Upper left: case of canine lymphoma. Upper right: case of canine osteosarcoma. Lower left: case 1 of canine renal cell carcinoma. Lower right: case 2 of canine renal cell carcinoma.

FIG. 5-2 Immunohistochemical staining images of other tumors. Left: case of canine squamous cell carcinoma. Right: case of canine fibrosarcoma.

FIG. 6 Immunohistochemical staining image of a case of ovine listeriosis. Left: PD-L1 staining image of a brain lesion of ovine listeriosis exhibiting neurologic symptoms. Right: enlarged photograph of the left image.

FIG. 7 Immunohistochemical staining images of porcine infections. Left: case of porcine circovirus type 2 infection. Right: case of porcine mycoplasma pneumonia.

FIG. 8 Alignment of amino acid sequences of the constant region of rat Ig kappa chain (light chain).

FIG. 9 Alignment of amino acid sequences of the constant region of rat IgG2a chain (heavy chain).

FIG. 10 Schematic drawing of pDC6 vector and a rat-human chimeric anti-PD-L1 antibody.

FIG. 11 Binding of rat anti-bovine PD-L1 antibodies 6C11-3A11 and 6G7-E1 to canine PD-L1-EGFP expressing cells. 6C11-3A11 specifically bound to canine PD-L1-EGFP expressing cells.

FIG. 12 Immunohistochemical staining images of skin squamous cell carcinoma, nasal adenocarcinoma and transitional cell carcinoma in dogs. No specific signals were detected with 6G7-E1. Tumor cells were stained with 6C11-3A11.

FIG. 13 Immunohistochemical staining images of anal sac gland carcinoma, soft tissue sarcoma and osteosarcoma in dogs. In anal sac gland carcinoma and soft tissue sarcoma, no specific signals were detected with 6G7-E1, but tumor cells were stained with 6C11-3A11. In osteosarcoma, both antibodies stained tumor cells, but stronger signals were obtained with 6C11-3A11.

FIG. 14 Immunohistochemical staining images of oral malignant melanoma, mammary adenocarcinoma, histiocytic sarcoma, diffuse large B-cell lymphoma and transmissible venereal tumor in dogs using 6C11-3A11. In the tumor species other than transmissible venereal tumor, PD-L1 on tumor cells was stained.

FIG. 15 Binding of rat anti-bovine PD-L1 antibody 6C11-3A11 to bovine PD-L1-EGFP expressing cells. 6C11-3A11 specifically bound to bovine PD-L1-EGFP expressing cells.

FIG. 16 Immunohistochemical staining images of ileal lesions of cattle naturally and experimentally infected with Mycobacterium avium subsp. paratuberculosis, using (a) 6C11-3A11 and (b) Ziehl-Neelsen staining. 6C11-3A11 detected PD-L1 expression in cells infected with M. avium subsp. paratuberculosis (positive in Ziehl-Neelsen staining).

BEST MODES FOR CARRYING OUT THE INVENTION

Hereinbelow, the present invention will be described in detail.

The present invention provides an anti-PD-L1 antibody comprising (a) a light chain comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5).

CDR1, CDR2 and CDR3 in the light chain variable region (VL) of rat anti-bovine PD-L1 antibody 6C11-3A11 (monoclonal antibody) established by the present inventors are a region consisting of the amino acid sequence of KSISKY (SEQ ID NO: 1), a region consisting of the amino acid sequence of SGS and a region consisting of the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2), respectively (see FIG. 2).

Further. CDR1, CDR2 and CDR3 in the heavy chain variable region (VH) of rat anti-bovine PD-L1 antibody 6C1-3A1 are a region consisting of the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), a region consisting of the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and a region consisting of the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5), respectively (see FIG. 2).

In the amino acid sequences of KSISKY (SEQ ID NO: 1), SGS and QQHNEYPLT (SEQ ID NO: 2), as well as the amino acid sequences of GYTFTDYI (SEQ ID NO: 3), INPDSGGN (SEQ ID NO: 4) and ARGITMMVVISHWKFDF (SEQ ID NO: 5), one, two, three, four or five amino acids may be deleted, substituted or added. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as CDR of VL or CDR of VH of the PD-L1 antibody.

As used herein, the term “antibody” is a concept encompassing not only full-length antibodies but also antibodies of smaller molecular sizes such as Fab, F(ab)′₂. ScFv, Diabody, V_(H), V_(L). Sc(Fv)₂, Bispecific sc(Fv)₂, Minibody, scFv-Fc monomer and scFv-Fc dimer.

The anti-PD-L1 antibody of the present invention may be derived from rat. For example, the anti-PD-L1 antibody may be a rat anti-bovine PD-L1 antibody.

The amino acid sequence of the VL and the amino acid sequence of the VH of rat anti-bovine PD-L1 antibody 6C11-3A11 (monoclonal antibody) are shown in SEQ ID NOS: 6 and 7, respectively. The amino acid sequences as shown in SEQ ID NOS: 6 and 7 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as VL or VH of the PD-L1 antibody.

There are two types of immunoglobulin light chain, which are called Kappa chain (κ) and Lambda chain (λ). In the anti-PD-L1 antibody of the present invention, the light chain constant region (CL) may have the amino acid sequence of the constant region of either Kappa chain or Lambda chain. However, the relative abundance of Lambda chain is higher in ovine, feline, canine and equine, and that of Kappa chain is higher in mouse, rat, human and porcine. Rat anti-bovine PD-L1 antibody 6C11-3A11 (monoclonal antibody) is a rat-derived IgG2a, and the CL thereof has the amino acid sequence of the constant region of Kappa chain.

The heavy chain constant region (CH) of the anti-PD-L1 antibody of the present invention may have the amino acid sequence of the constant region of rat IgG2a. Immunoglobulin heavy chain is classified into γ chain, μ chain, α chain, δ chain and ε chain depending on the difference in constant region. According to the type of heavy chain present, five classes (isotypes) of immunoglobulin are formed, they are IgG, IgM, IgA, IgD and IgE.

Immunoglobulin G (IgG) accounts for 70-75% of human immunoglobulins and is the most abundantly found monomeric antibody in plasma. IgG has a four-chain structure consisting of two light chains and two heavy chains. Human IgG1, IgG2 and IgG4 have molecular weights of about 146,000, whereas human IgG3 has a long hinge region that connects Fab region and Fc region and has a larger molecular weight of 170,000. Human IgG1 accounts for about 65%, human IgG2 about 25%, human IgG3 about 7%, and human IgG4 about 3% of human IgG. They are uniformly distributed inside and outside of blood vessels. Having a strong affinity for Fc receptors and complement factors on effector cell surfaces, human IgG1 induces antibody-dependent cell cytotoxicity (ADCC) and also activates complements to induce complement-dependent cell cytotoxicity (CDC). Human IgG2 and IgG4 are low at ADCC and CDC activities because their affinity for Fc receptors and complement factors is low.

Immunoglobulin M (IgM), which accounts for about 10% of human immunoglobulins, is a pentameric antibody consisting of five basic four-chain structures joined together. It has a molecular weight of 970,000. Usually occurring only in blood, IgM is produced against infectious microorganisms and takes charge of early stage immunity.

Immunoglobulin A (IgA) accounts for 10-15% of human immunoglobulins. It has a molecular weight of 160,000. Secreted IgA is a dimeric antibody consisting of two IgA molecules joined together. IgA1 is found in serum, nasal discharge, saliva and breast milk. In intestinal juice, IgA2 is found abundantly.

Immunoglobulin D (IgD) is a monomeric antibody accounting for no more than 1% of human immunoglobulins. IgD is found on B cell surfaces and involved in induction of antibody production.

Immunoglobulin E (IgE) is a monomeric antibody that occurs in an extremely small amount, accounting for only 0.001% or less of human immunoglobulins. Immunoglobulin E is considered to be involved in immune response to parasites but in advanced countries where parasites are rare. IgE is largely involved in bronchial asthma and allergy among other things.

With respect to rat, sequences of IgG1, IgG2a, IgG2b and IgG2c have been identified as the heavy chain of IgG. Rat anti-bovine PD-L1 antibody 6C11-3A11 has the amino acid sequence of the CH of IgG2a.

In the antibody of the present invention, it is more preferable that the CL has the amino acid sequence of the constant region of Kappa chain and that the CH has the amino acid sequence of the constant region of IgG2a.

The amino acid sequence and the nucleotide sequence of the VL of rat anti-bovine PD-L1 antibody 6C11-3A1 identified by the present inventors are shown in SEQ ID NOS: 6 and 14, respectively.

The amino acid sequence and the nucleotide sequence of the VH of rat anti-bovine PD-L1 antibody 6C11-3A11 identified by the present inventors are shown in SEQ ID NOS: 7 and 15, respectively.

The amino acid sequence and the nucleotide sequence of the CL (Kappa chain) of rat anti-bovine PD-L1 antibody 6C11-3A11 identified by the present inventors are shown in SEQ ID NOS: 8 and 16, respectively. These sequences are identical with the sequences registered at GenBank (a nucleotide sequence database provided by National Center for Biotechnology Information (NCBI)) under accession numbers #XM_008775358.2. #BC062802.1, #3C088255.1, #L22653.1. #L22655.1 and #M14434.1.

The amino acid sequence and the nucleotide sequence of the CH (IgG2a) of rat anti-bovine PD-L1 antibody 6C11-3A11 identified by the present inventors are shown in SEQ ID NOS: 9 and 17, respectively. These sequences are identical with the sequences registered at GenBank under accession numbers #BC088240.1. #BC091257.1. #BC091272.1. #BC088423.1, #L22652.1 and #L22654.1.

Amino acid sequences and nucleotide sequences of CLs and CHs for rat antibodies other than the above may be obtained from known databases for use in the present invention.

As an amino acid sequence and a nucleotide sequence of rat Ig Kappa chain, the sequence registered at GenBank under accession number #V01241.1 is shown in SEQ ID NOS: 10 and 18.

As an amino acid sequence and a nucleotide sequence of rat Ig Kappa chain, the sequence registered at GenBank under accession number #X16129.1 is shown in SEQ ID NOS: 11 and 19.

As an amino acid sequence and a nucleotide sequence of rat Ig Kappa chain, the sequence registered at GenBank under accession number #DQ402471.1 is shown in SEQ ID NOS: 12 and 20.

As the CH of rat IgG2a, the sequence registered at GenBank under accession number #DQ402472.1 is shown in SEQ ID NOS: 13 and 21.

The anti-PD-L1 antibody of the present invention may be an anti-PD-L1 antibody in which the CL has the amino acid sequence as shown in any one of SEQ ID NOS: 8 and 10 to 12 and the CH has the amino acid sequence as shown in SEQ ID NO: 9 or 13.

The amino acid sequences as shown in SEQ ID NOS: 8 to 13 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as CL or CH of the PD-L antibody.

Alignments of amino acid sequences of the CL and the CH of a rat anti-PD-L1 antibody are shown in FIG. 8 and FIG. 9, respectively. The above-described mutations such as deletion, substitution or addition of amino acids may suitably have occurred at the mutation sites as shown in FIGS. 8 and 9 or at the vicinity thereof.

The anti-PD-L1 antibody of the present invention may be a chimeric antibody. The VL and the VH of the antibody may be suitably derived from rat. For example, the VL may be the VL of a rat anti-PD-L1 antibody (e.g., 6C11-3A11); the VH may be the VH of a rat anti-PD-L1 antibody, and the CL and the CH may be derived from an animal other than rat. For example, when a rat antibody is chimerized using the constant regions of a mouse antibody, the resulting chimeric antibody will be useful for testing and diagnosis because various secondary antibodies to mouse antibodies are commercially available. Amino acid sequences and nucleotide sequences of the CLs and the CHs of antibodies of animals other than rat may be obtained from known databases for use in the present invention.

Amino acid sequences and nucleotide sequences of CLs and CHs for human, mouse, bovine, canine, ovine, porcine and water buffalo are summarized in the table below.

TABLE GenBank Accession Species Ig Domain Nucleotide Sequence Amino Acid Sequence No. IMGT Database Reference Human Human IgG4 GCACCTGAGTTCCTGGCGGGAOCATCAGTCTTCCTC KPKDTLMISRTPEVTCVCCDVSQEDPE K01316 http://www. Ellison (Scientific Ig variant TTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC VQFNWYVDCVEVHNAKTKPREEGFNS imgt.org/ J. et al Name: heavy 1 GGGACCGCTGAGGTCACGTGCGTGGTGGTGGACGTG TYRWSVLTVLHQDWLNGKEYKCKVS IMGT DNA, 1 Homo chain AGGCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC NKGLPSSIEKTISKAKGQPREPQVYTLP reportoire/ 11-16 sapiens) re- GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAG PSGEEMTKNQVSLTCLVKGFYPSDIAV index. (1981) gion CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG EWESNGQPENNYKTTPPVLDSDGSFFL ghp?section PMID: (CH1 GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG YSRLTVDKSRWQEGNVFSCSVMHEAL =locus 6299662 CH3) AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA HNHYTQKSLSLSLGK* Genes& GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA (SEQ ID NO: 30) reportoire= GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC genetable& CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG species= GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC human& AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG gcf=IGHC CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTA ACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTC TTCTCATGCTCCGTGARGCATGAGGCTCTGCACAAC CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGT AAATGA (SEQ ID NO: 31) IgG4 GAGTCCAAATATGGTCCCCCGTGGCCATCATGGCCA ESKYGPPCPSCPAPEFLGGPSVFLFPP AJ001583 Brusco variant GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTG KPKDTLMISRTPEVTCVVVDVSGEDPE A. et al. 2 TTCCCCCCAAAACCCAAGGACACTCTGATGATCTCC VQFNWYVDGVEVHNAKTKPREEQFNS Eur. J. CGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG TYRVVSVLTVVHQDWLNGKEYKCKVS Immuno AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC NKGLPSSIEKTISKAKGQPREPQVYTLP genol., GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAG PSQEEMTKNQVSLTCLVKGFYPSDIAV 25, CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG EWESNGQPENNYKTTPPVLDSDGSFFL 348-355  GTCAGCGTGCTCACGGTCGTGCACCAGGACTGGCTG YSRLTVDKSRWQEGNVFSCSVMHEAL (1998). AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA HNHYTQKSLSLSLGK* PMID: GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA (SEQ ID NO: 32) 9805657 GCCAAAGGCCACCCCCGAGAGCCACAGCTGTACACC CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG GTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGOTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTA ACGGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCTCTGGGT AAATGA (SEQ ID NO: 33) IgG4 GCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTG APEFLGGPSVFLFPPKPKDTLMISRTPE AJ001564 variant TTCCCCCCAAAACCCAAGGACACTCTCATGATCTCC VTCVVVDVSQEDPEVQFNWYVDGVEV 3 CGGACCCCTGAGGTCACGTCCGTGGTGGTGGACGTG HNAKTKPREEQFNSTYRVVSVLTVLHQ AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC DWLNGKEYKCKVSNKGLPSSIEKTISKA GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAG KGQPREPQVYTIPPSQEEMTKNQVSL CCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG TCLVKGFYPSDIAVEWESNGQPENNYK GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTG TTPPVLDSDGSFFLYSKLTVDKSRWQE AACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAA GNVFSCSVMHEALHNHYTQKSLSLSLG GGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAA K* GCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACC (SEQ ID NO 34) CTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAG GTCAGCCTGACCTGCGTGGTCAAAGGCTTCTACCCC AGCGACATCGCGGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTC ACCGTGGACAAGAGCAGGTGGCAGGAGGGGAACGTC TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC CACTACACGCAGAAGAGCCTCTCCCTGTCTCTGGGT AAATGA (SEQ ID NO: 33) Human Ig ACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA TVAAPSVFIFPPSDEQLKSGTASVVCL X96754 http://www. GL Ig kappa TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT LNNFYPRCAKVQWKVDNALQSGNSQE imgt.org/ light (CK) GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC SVTEQDSKDSTYSLSSTLTLSKAQYEK IMGT con- AAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG HKVYACEVTHQCLSSPVTKSFNRGEC* reportoire/ stant GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGG (SEQ ID NO: 28) index. region AAGGACAGCACCTACAGCCTCAGCAGGACCCTGACG ghp?section CTGAGCAAAGCAGACTACGAGAAACACAAAGTCTAC =locus GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCC Genes& GTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG reportoire= (SEQ ID NO: 29) genetable& species= human& gcf=IGHC Mouse Mouse IgG1 GCCAAAACGACACCCCCATCTGTCTATCCACTGGCCC AKTTPPSVYPLAPGSAAQTNSMVTLG J00453 http://www. Honjo T. (Scientific Ig variant CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCT CLVKGYFPEPVTVTWNSGSLSSGVHTF AH005309 imgt.org/ et al. Name: Mus heavy 1 GGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGT PAVLESDLYTLSSSVTVPSSPRPSETV V00793 IMGT Ceit Musculus) chain GACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGG TCNVAHPASSTKVDKKIVPRDCGCKPC D78344 reportoire/ 18, 558- region TGTGCACACCTTCCCAGCTGTCCTGGAGTCTGACCTC ICTVPEVSSVFIFPPKPKDVLTITLTPK index. 568 (1979) (CH1 TACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCC VTCVVVDISKDDPEVQFSWFVDDVEVHT ghp?section PMID: CH3) CTCGGCCCAGCGAGACCGTCACCTGCAACGTTGCCC AQTQPREEQFNSTFRSVSELPIMHQDW =locus 115593 ACCCGGCGACCAGCACCAAGGTGGACAAGAAAATTG LNGKEFKCRVNSAAFPAPIEKTISKTKG Genes& Akohoni Y. TGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTAC RPKAPQVYTIPPPKEQMAKDKVSLTCM reportoire= and AGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCA ITDFFPEDITVEWQWNGQPAENYKNTQ genetable& Kursawa Y. AAGCCCAAGGATGTGCTCACCATTACTCTCACTCCTA PIMNTNGSYFVYSKLNVQKSNWEAGNT species= Genomes, AGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGA FTCSVLHEGLHNHHTEKSLSHSPGK human& 41, 100- TCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTG (SEQ ID NO: 44) gcf=IGHC 104 (1997) GAGGTGCACAGAGCTCAGACGCAAGCGCGGGAGGAG PMID: GAGTTCAACAGCACTTTGGGCTCAGTCAGTGAACTTC 9126488 CCATCATGCACCACGACTGCCTCAATCGCAAGCAGTT CAAATGCAGGGTCAACAGTCCAGCTTTCCCTGCCCCC ATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCG AAGGCTCCACAGGTGTACAGCATTCCACCTCCCAAGG AGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCAT GATAACAGACTTCTTCCCTGAAGACATTACTGTGGAG TCGCACTGGAATGCGCAGCCAGCCGAGAACTACAAG AACACTCAGCCCATCATGAACACGAATGGCTCTTACT TCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTG GGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACAT GAGGGCCTGCACAACCACCATACTGAGAAGAGCCTC TCCCACTCTCCTGGTAAATGA (SEQ ID NO: 45) IgG1 GCCAAAAGGACACCCCCATCTGTCTATCCACTGGCCC AKTTPPSVYPLAPGSAAQTNSMVTLG L35252 Honjo T. variant CTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCT CLVKGYFPEPVTVTWNSGSLSSGVHTF et al, 2 GGCATGCCTGGTCAAGGGCTATTTCCCTGACCCAGT PAVLQSDLYTLSSSVTVPSSTWSQTV Ceit, 18 GACACTGACCTGGAACTCTGGATCCCTGTCCAGCGG TCNVAHPASSTKVDKKIVPRDCGCKPC 558-568 TGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTC ICTVPEVSSVFIFPPKPKDVLTITLTPK (1979) TACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCA VTCVVVDISKDDPEVQFSWFVDDVEVHT PMID: CCTGGCCCAGCCAGACCGTCACCTGCAACGTTGCCC AQTKPREEQINSTFRSVSELPIMHQDWL 115593 ACCCGGCCAGCAGCACCAAGGTCGACAAGAAAATTG NGKEFKCRVNSAAFPAPIEKTISKTKGR TGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTAC PKAPQVYTIPPPKEQMAKDKVSLTCMI AGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCA TNFFPEDITVEWQNGQPAENYKNTQP AAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTA IMDTDGSYFVYSKLNVQKSNWEAGNTF AGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGA TCSVLHEGLHNHHTEKSLSHSPGK TCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTG (SEQ ID NO: 46) GAGGTGCACACAGCTCAGACGAAACCCCGGGAGGAG CAGATCAACAGCACTTTCCGTTCAGTCAGTGAACTTC CCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTT CAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCC ATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCG AAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGG AGCAGATGGCCAAGGATAAAGTGAGTGTGACCTGCAT GATAACAAACTTGTTCCCTGAAGAGATTACTGTGGAG TGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAG AACACTCAGCCCATCATGGACACAGATGGCTCTTACT TTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTG GGAGGCAGGAAATACTTTCACCTCCTCTGTGTTACAT TGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTC TCCCACTCTCCTGGTAAATGA (SEQ ID NO: 47) IgG2a GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCC AKTTAPSVYPLAPVCGDTTGSSVTLGC J00470 Yamawaki variant CCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTC LVKGYFPEPVTLTWNSGSLSSGVHTFP AH003509 Kataska 1 TAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGT AVLQSDLYTLSSSVTVTSSTWPSQSIT V00825 Y. et al, GACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGT CNVAHPASSTKVDKKIEPRGPTIKPCPP V00766 Nucleic GTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCT CKCPAPNLLGGPSVFFPPKIKDVLMISL D78344 Acids ACACCCTCAGCAGCTCAGTGACTGTAAGCTCGAGCAC SPIVTCVVVDVSEDDPDVQISWFVNNV Res. 9, CTCGCCCAGCCAGTCCATCACCTGCAATGTGGCCCA EVHTAQTQTHREDYNSTLRVVSALPIQ 1365-1381 CCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGA HQDWMSGKEFKCKVNNKDLPAPIERTI (1981). GCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATG SKPKGSVRAPQVYVLPPPEEEMTKKQV PMID: CAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCC TLTCMVTDFMPEDIYVEWTNNGKTELN 6262729 GTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCA YKNTEPVLDSDGSYFMYSKLRVEKKNE Oto F. et TGATCTCCCTGAGCCCCATAGTCACATCTGTGGTGGT VERNSYSCSVVHEGLHNHHTTKSFSRT al, Proc GGATGTGAGCGAGGATGACCCAGATGTCCAGATGAG PGK Natl. CTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAG (SEQ ID NO: 48) Acad Sci. ACACAAACCCATAGAGAGGATTACAACAGTACTCTCC U.S.A, 78, GGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACT 2442-2446 GGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACA (1981), ACAAAGACCTCCCAGCGCCCATGGAGAGAACCATCT PMID: CAAAACCCAAAGGGTGAGTAAGAGGTCCACAGGTATA 0787604 TGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAA Sikorev J. CAGGTCACTCTGACCTGCATGGTCACAGACTTCATGC et al, CTGAAGACATTTACGTGGAGTGGACCAACAACGGGA Nucleic AAACAGAGCTAAACTACAAGAACACTGAACCAGTCCT Acids Res. GGACTCTGATGGTTCTTACTTCAFGTACAGCAAGCTG 8, 3143- AGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGC 3155 (1980) TACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATC PMID: ACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTA 6777755 AATGA Akahon Y. (SEQ ID NO: 49) and Kurwaska Y. Genomic, 41, 100- 104 (1997) PMID: 9128488 IgG2a GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCC AKTTAPSVYPLAPVCGDTTGSSVTLGC X16997 Morgado variant CCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTC LVKGYFPEPVTLTWNSGSLSSGVHTFP M.G. et 2 TAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGT AVLQSDLYTLSSSVTVTSSTWPSQSIT al, EMBO GACCTTGACCTGGAACTCTGCATCCCTGTCCAGTGGT CNVAHPASSTKVDKKIEPRGPTIKPCPP J. S. GTACACACCTTCCCAGCTGTCCTGCAGTCTGACCTCT CKCPAPNLLGGPSVFIFPPKIKDVLMIS 3245- ACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCAC LSPMVTCVVVDVSEDDPDVQISWFVNN 3251  CTGGCCGACCCAGTCGATCACCTGCAATGTGGCCCA VEVLTAQTQTHREDYNSTLRVVSALPI (1989) CCGGGCAAGCAGCACCAAGGTGGACAAGAAAATTGA QHQDWMSGKEFKCKVNNKALPAPIERT PMID: GCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATG ISKPKGSVRAPQVYVLPPPEEEMTKKQ 2510996 CAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCC VTLTCMVTDFMPEDIYVEWTNNGKTEL GTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCA NYKNTEPVLDSDGSYFMYSKLRVEKKN TGATCTCCCTGACTCCCATGCTCACATGTGTGGTGGT WVERNSYSCSVVHEGLHNHHTTKSFSR GGATGTGAGCGAGGATGACCCAGATGTCCAGATCAG TPGK CTGGTTCGTGAACAACGTGGAAGTACTCACAGCTCAG (SEQ ID NO: 50) ACACAAACCCATAGAGAGGATTACAACAGTACTCTCC GGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACT TGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACA ACAAAGCCCTCCCAGCGCCCATCGAGAGAACCATCT TCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATA TGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAA CAGGTCACTCTGACCTGCATGGTCACAGACTTCATGC CTGAAGACATTTACGTGGAGTGGACCAACAACGGGA AAACAGAGCTAAACTACAAGAACACTCAACCAGTCCT GGACTCTGATGGTTCTTACTTCATGTACACCAAGCTG AGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGC TACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATC ACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTA AATGA (SEQ ID NO: 51) IgG2b GCCAAAACAACACCCCCATCAGTCTATCGACTGGCCC AKTTPPSVYPLAPGCGDTTGSSVYLGG J00461 Yamawaki variant CTGGGTGTGGAGATACAACTGGTTCCTCCGTGACTCT LVKGYFPESVTVTVWSGSLSSSVHTFP AH005300 Kataoka Y. 1 GGGATGCCTGGTCAAGGGCTACTTCGCTGAGTCAGT ALLQSGLYTMSSSVTVPSSTWPSQTVT V00801 et al, GACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGT CSVAHPASSTTVDKKLEPSGPISTINPC D78344 Nature, GTGCACACCTTCCGAGCTCTCCTGCAGTCTGGACTCT PPCKECHKCPAPNLEGGPSVFIFPPNIK 283, ACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCAC DVLMISLTPKVTCVVVDVSEDDPDVQI 786-789 TTTGGCCAAGTCAGACCGTCACCTGCAGCGTTGCTCAC SWFVNNVEVHTAQTQTHREDYMSTIRV (1980) CGAGGCAGCAGCACCACGGTGGACAAAAAACTTGAG VSTLPIQHQDWMSGKEFKCKVNNKDLP PMID: CCCAGCGGGCCCATTTCAAGAATCAACGCCTGTCCTC SPIERTISKIKGLVRAPQVYILPPPAEQ 6766534 GATCCAAGGAGTGTCACAAATGCCCAGCTCCTAACCT LSRKDVSLTCLVVGFNPGDISVEWTSNG Oilo R. CGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAT HTEENYKDTAPVLDSDGSYFIYSKLNM and ATCAAGGATGTACTCATGATCTCCCTGACACCCAAGG KTSKWEKTDSFSCNVRHEGLKNYYLKK Raugeon F. TCACGTGTGTGGTGGTGGATGTGAGCGAGGATGACC TISRSPGK Nature, CAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGA (SEQ ID NO: 52) 298, 761- AGTACACACAGCTCAGACACAAACCCATAGAGAGGAT 763 (1982) TACAACAGTACTATCCGGGTGGTCAGCACCCTCCGCA PMID: TCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCA 6803173 AATGCAAGGTCAACAACAAAGACCTCCCATCACCCAT Akahon Y CGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCAGA and GCTCCACAAGTATACATCTTGCCGCCACCAGCAGAGC Kurosawa AGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTGGT Y CGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGTG Genomics, GACCAGCAATGGGCATACAGAGGAGAACTACAAGGA 41, 100- CACCGCACCAGTCCTAGACTCTGACGGTTCTTACTTC 104 (1997) ATATATAGCAAGCTCAATATGAAAACAAGCAAGTGGG PMID: AGAAAACAGATTCCTTCTCATGCAACGTGAGACACGA 9126488 GGGTCTGAAAAATTACTACCTGAAGAAGACCATCTCC CGGTCTCCGGGTAAATGA (SEQ ID NO: 53) IgG2b GCCAAAACAACACCCCCATCAGTCTATCCACTGGCCC AKTTPPSVYPLAPGCGDTTGSSVTSG V00763 Tuckor variant CTGGGTGTGGAGATAGAACTGGTTCCTCCGTGACCTC CLVKGYFPEPVTVTWNSGSISSSVHTF P.W. et 2 TGGGTGCCTGGTCAAGGGGTACTTCCCTGAGCCAGT PALLQSGLYTMSSSVTVPSSTWPSQTV al, GACTGTGACTTGGAACTCTGGATCCCTGTCCAGCAGT TCSVAHPASSTTVDKKLEPSGPISTINP Science GTGCACACCTTCCCAGCTCTCCTGCAGTCTGGACTCT CPPCKECHKCPAPNLEGGPSVFIFPPNI 206 ACACTATGAGCAGCTCAGTGACTGTCCCCTCCAGCAC KDVLMISLTPKVTCVVVDVSEDDPDVQ 1303-1306 CTGGCCAAGTCAGACCGTCACCTGCAGCGTTGGTCA ISWFVNNVEVHTAQTQTHREDYNSTIRV (1979) CCCAGCCAGCAGCACCACGGTGGACAAAAAACTTGA VSTLPIQHQDWMSGKEFKCKVNNKDLP PMID GCCCAGCGGGCCCATTTCAACAATCAACCCCTGTCCT SPIERTISKIKGLVRAPQVYTLPPPAEQ 117549 CCATGCAAGGAGTGTCACAAATGCCCAGCTCCTAACC LSRKDVSLTCLVVGFNPGDISVEWTSNG TCGAGGGTGGACCATCCGTCTTCATCTTCCCTCCAAA HTEENYKDTAPVLDSDGSYFIYSKLNM TATCAAGGATGTACTCATGATCTCCCTGACACCCAAG KTSKWEKTDSFSCNVRHEGLKNYYLKK GTCACGTGTGTGGTGGTGGATGTGAGCGAGGATGAC TISRSPGK CCAGACGTCCAGATCAGCTGGTTTGTGAAGAACGTGG (SEQ ID NO: 54) AAGTACACACAGCTCAGACACAAACCCATAGAGAGG ATTACAACAGTACTATCCGGGTGGTGAGCACCCTCCC CATCCAGCACCAGGACTGCATGAGTGGCAAGGAGTT CAAATGCAAGGTGAACAACAAAGACCTCCCATCACCC ATCGAGAGAACCATCTCAAAAATTAAAGGGCTAGTCA GAGCTCCACAAGTATACACTTTGCCGCCACCAGCAGA GCAGTTGTCCAGGAAAGATGTCAGTCTCACTTGCCTG GTCGTGGGCTTCAACCCTGGAGACATCAGTGTGGAGT GGACCACCAATGGGCATACAGAGGAGAACTACAAGG ACACCGCACCAGTTCTTGACTCTGACGGTTCTTACTT CATATATAGCAAGCTCAATATGAAAACAAGCAAGTGG GAGAAAAGAGATTCCTTCTCATGCAACGTGAGACACG AGGGTCTGAAAAATTACTACCTGAAGAAGACCATCTC CCGGTCTCCGGGTAAATGA (SEQ ID NO: 55) IgG2c GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCC AKTTAPSVYPLAPVCGGTTGSSVTLGC J00479 Olio R. variant CCTGTGTGTGGAGGTACAACTGGCTCCTCGGTCACTC LVKGYFPEPVTLTWNSGSLSSGVHTFP and 1 TAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGT ALLQSGLYTLSSSVTVTSNTWPSQTIT Rougeon F. GACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGT CNVAHPASSTKVDKKIEPRVPITQNPCP Cell, 32 GTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCGTCT PLKECPPCAAPDLLGGPSVFIFPPKIKD 515-523 ACACCCTCAGCAGCTCAGTGACTGTAACCTCGAACAC VLMISLSPMVTCVVVDVSEDDPDVQIS (1983). CTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCA WFVNNVEVHTAQTQTHREDYNSTLRV PMID: CCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGA VSALPIQHQDWMSGKEFKCKVNNRALP 6297797 GCCCAGAGTGCCCATAACACAGAACCGGTGTCCTCC SPIEKTISKPRGPVRAPQVYVLPPPAEE ACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCT MTKKEFSLTCMITGFLPAEIAVDWTSNG CTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAG RTEQNYKNTATVLDSDGSYFMYSKLRV ATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGG QKSTWERGSLFACSVVHEVLHNHLTTK TCACATGTGTGGTGGTGGATGTGAGCGAGGATGACC TISRSLGK CAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGA (SEQ ID NO: 56) AGTACACACAGCTCAGACACAAACCCATAGAGAGGAT TACAACAGTACTCTCCGGGTGGTGAGTGCCCTCCCCA TCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCA AATGCAAGGTCAACAACAGAGCCCTCCCATCCCCCAT CGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAG AGCTCCACAGGTATATGTCTTGGCTCCACCAGCAGAA GAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGA TCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTG GACCAGCAATGGGCGTACAGAGGAAAACTACAAGAA CACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTC ATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGG AAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGA GGTGCTGCACAATCACCTTACGACTAAGACCATCTCC CGGTCTCTGGGTAAATGA (SEQ ID NO: 57) IgG2c GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCC AKTTAPSVYPLAPVCGGTTGSSVTLGC X16998 Morgado variant CCTGTGTGTGGAGGTACAACTGGCTCCTCGGTGACTC LVKGYFPEPVTLTWNSGSLSSGVHTFP M.G. et 2 TAGGATGCCTGGTCAACGGTTATTTCCCTGAGCCAGT ALLGSGLYTLSSSVTVTSNTWPSQTIT al., EMBO GACCTTGACCTGGAACTCTGGATCGCTGTCCAGTGGT CNVAHPASSTKVDKKIESRRPIPPNSCP J. 8, GTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCCTCT PCKECSIFPAPDLLGGPSVFIFPPKIKD 3245-3251 ACACCCTGAGCAGCTCAGTGACTGTAACCTCGAACAC VLMISLSPIVTCVVVDVSEDDPDVQISW (1989). CTGGCCCAGCCAGACCATCACCTGCAATGTGGCGCA FVNNVEVHTAQTQTHREDYNSTLRVVS PMID: CCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGA ALPIQHQDWMSGKEFKCKVNNRALPSP 2510996 ATCCAGAAGGCCCATACCACCCAACTCCTGTCCTCCA IEKTISKPRGPVRAPQVYVLPPPAEEMT TGCAAAGAGTGTTCCATATTCCCAGCTGCTGACCTCT KKEFSLTCMITDFLPAEIAVDWTSMGHK TGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGAT ELNYKNTAPVLDTDGSYFMYSKLRVQK CAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTC STWEKGSLFACSVVHEGLHNHHTTKTI ACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCA SRSLGK GATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAG (SEQ ID NO: 58) TACACACAGCTCAGACACAAACCCATAGAGAGGATTA CAACAGTACTCTCCCGGTGGTCAGTGCCCTCCCCATC CAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAA TGCAAGGTCAACAACAGAGCCCTCCCATCCCCCATC GAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAGA GCTCCACAGGTATATGTCTTGCGTCGACGAGCAGAAG AGATGACTAAGAAAGAGTTCAGTCTGAGCTGCATGAT CACAGACTTCTTACCTGCCGAAATTGCTGTGGACTGG ACCAGCAATGGGCATAAAGAGCTGAACTACAAGAACA CCGCACCAGTCCTGGACACTGATGGTTCTTACTTCAT GTACAGCAAGCTCAGAGTGCAAAAGAGCACTTGGGA AAAAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGAG GGTCTGCACAATCACCATACGACTAAGACCATCTCCG GGTCTCTGGGTAAATGA (SEQ ID NO: 59) IgG2c GCCAAAACAACAGCCCCATCGGTCTATCCACTGGCC AKTTAPSVYPLAPVCGGTTGSSVTLGC Y10606 Martin R.M variant CCTGTGTGTCGAGGTACAACTGGCTCCTCGGTGACTC LVKGYFPEPVTLTWNSGSLSSGVHTFP et al, 3 TAGGATGCCTGGTCAAGGGTTATTTCGCTGAGCCAGT ALLQSGLYTLSSSVTVTSNTWPSQTIT Immuno- GACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGT CNVAHPASSTKVDKKIEPRVPITGNPCP genics, 46, GTGCACACCTTCCCAGCTCTCCTGCAGTCTGGCCTCT PLKECPPCAAPDLLGGPSVFIFPPKIKD 157-168 ACAGGCTCAGCAGCTCAGTGACTGTAACCTCGAACAC VLMISLSPMVTCVVVDVSEDDPDVQIS (1997). CTGGCCCAGCCAGACCATCACCTGCAATGTGGCCCA WFVNNVEVHTAQTQTHREDYNSTLRV PMID: CCCGGCAAGCAGCACCAAAGTGGACAAGAAAATTGA VSALPIQHQDWMSGKEFKCKVNNRALP 9162106 GGCCAGAGTGCCCATAACACAGAACCCCTGTCCTCC SPIEKTISKPRGPVRAPQVYVLPPPAEE ACTCAAAGAGTGTCCCCCATGCGCAGCTCCAGACCT MTKKEFSLTQMITGFLPAEIAVDWTSNG CTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAG RTEQNVKNTATVLDSDGSYFMYSKLRV ATCAAGGATGTACTCATGATCTCCCTGAGCCCCATGG QKSTWERGSLFACSVVHEGLHNHLTTK TCACATGTGTGCTGCTGGATGTGAGCGAGGATGACC TISRSLGK CAGACGTCCAGATCAGCTGGTTTGTGAACAACGTGGA (SEQ ID NO: 60) AGTACACACAGCTCAGACAGAAACCCATAGAGAGGAT TACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCA TCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCA AATGCAAGGTCAACAACAGAGCCCTCCCATCCCGCAT CGAGAAAACCATCTCAAAACCCAGAGGGCCAGTAAG AGCTCCACAGGTATATGTCTTGGCTCCACCAGCAGAA GAGATGACTAAGAAAGAGTTCAGTCTGACCTGCATGA TCACAGGCTTCTTACCTGCCGAAATTGCTGTGGACTG GACCAGCAATGCGCGTACAGAGCAAAACTACAAGAA CACCGCAACAGTCCTGGACTCTGATGGTTCTTACTTC ATGTACAGCAAGCTCAGAGTACAAAAGAGCACTTGGG AAAGAGGAAGTCTTTTCGCCTGCTCAGTGGTCCACGA GGGTCTGCACAATCACCTTACGACTAAGACCATCTCC CGGTCTCTGGGTAAATGA (SEQ ID NO: 61) IgG3 GCTACAACAACAGCCCCATCTGTCTATCCCTTGGTCC ATTTAPSVYPLVPGCSDTSGSSVTLGC J00451 Stanton CTGGCTGCAGTGACACATCTGGATGCTCGGTGACACT LVKGYFPEPVTVKVNWYGALSSGVRTVS AH005309 L.W. GGGATGCCTTGTCAAAGGCTACTTCCCTGAGCCGGTA SVLQSGFYSLSSLVTVPSSTWPSQTVI X00915 and Marcus ACTGTAAAATGGAACTATGGAGCCCTGTCCAGCGGTG CNVAHPASKTELIKRIEPRIPKPSTPPG D78343 K.B. TGCGCACAGTCTCATCTGTCCTGCAGTCTGGGTTCTA SSCPPGNILGGPSVFIFPPKPKDALMIS Nucleic TTGCGTCAGCAGCTTGGTGACTGTACCCTCCAGCACC LTPKVTCVVVDVSEDDPDVHVSWFVDN Acids Res. TGGCCCAGCCAGACTGTCATCTGCAACGTAGCCCAC KEVHTAWTQPREAQYNSTFRVVSALPI 10, 5993- CCAGCGAGCAAGACTGAGTTGATCAAGAGAATCGAG QHQDWMRGKFFKCKVNNKALPAPIERT 6006 (1982) CCTAGAATACCCAAGCCCAGTACGCCCCCAGGTTCTT ISKPKGRAQTPQVYTIPPPREQMSKKK PMID: CATGCCCACCTGGTAACATCTTGGGTGGACCATCCGT VSLTCLVTNFFSEAISVEWWRNGELEQD 629864 CTTCATCTTCCCCCCAAAGCCCAAGGATGCACTCATG YKNTPPILDSDGTYFLYSKLTVDTDSWL Wels J.A. ATCTCCCTAACCCCCAAGGTTACGTGTGTGGTGGTGG QGEIFTCSVVHEALHNHHTQKNLSRSP et al, ATGTGAGCGAGGATGACCCAGATGTCCATGTCAGCTG GK EMBO J. GTTTGTGGACAACAAAGAAGTACACACAGCCTGGACA (SEQ ID NO: 62) 2041-2046 CAGCCGCGTGAAGGTCAGTACAACAGTACCTTCCGA (1984) GTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGG PMID: ATCAGGGGCAAGGAGTTCAAATGCAAGGTCAACAAC 6092053 AAAGCCCTCCCAGCCCCCATCGAGAGAACCATCTCA Akahoni Y. AAACCCAAAGGAAGAGCCCAGACACCTCAAGTATAC and ACCATACCCCCACCTCGTGAACAAATGTCCAAGAAGA Kurosawa AGGTTAGTCTGACCTGCCTGGTCACCAACTTCTTCTC Y. TGAAGCCATCAGTGTGGAGTGGGAAAGGAACGGAGA Genomics, ACTGGAGCAGGATTACAAGAACAGTCCAGCCATCCTG 41, 100- GACTCAGATGGGACCTACTTCCTCTACAGCAACCTCA 10 (1997) CTGTGGATACAGACAGTTGGTTGCAAGGAGAAATTTT PMID: TACCTGCTCCGTGGTGCATGAGGCTCTCCATAACCAC 9126498 CACACACAGAAGAACCTGTCTCGCTGCCCTGGTAAAT GA (SEQ ID NO: 63) Mouse Ig GCTGATGCTGGACCAACTGTATCCATCTTCCCACCAT ADAAPTVSIFPPSSEQLTSGGASVVCF V00807 http://www. Hieter Ig kappa CCAGTGACCAGTTAACATCTGCACGTGCCTCAGTCGT LNNFYPKDINVKWKIDGSERQNGVLNS V00777 imgt.org/ P.A. et light (CK) GTGCTTCTTGAAGAACTTCTACCCCAAAGACATCAAT WTDQDSKDSTYSMSSTLTLTKDEYERH V01569 IMGT al. Cell, con- GTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATG NSYTCEATHKTSTSPIVKSFNRNEC V00806 reportoire/ 22, 197- stant GCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAG (SEQ ID NO: 36) X67002 index. 207 (1980) region ACAGCACCTACAGCATGAGCAGCACCCTCACGTTGA X67003 ghp?section PMID: CCAAGGACGAGTATGAACGAGATAACAGCTATACCTG X67004 =locus 6775818 TGAGGCCACTCACAAGACATCAACTTCACCCATTGTC X67005 Genes& Max E.E AAGAGCTTCAACAGGAATGAGTGTTAC X67006 reportoire= et al. J. (SEQ ID NO: 37) X67007 genetable& Bio. Chem, X67008 species= 256, 5116- X67009 mus musculus& 5120 (1981) X670010 group=IGKC PMID: X670011 6262318 X670012 Seidman J.G. et al, Nature, 280, 370- 375 (1979) PMID: 111146 Solin M.L. and Kaartnen M. Immuno- genics, 37 401-407 (1983) PMID: 8436414 Ig GGCCAGCCCAAGTCTTCGCCATCAGTCACCCTGTTTC GQPKSSPSVTLFPFSSEELETNKATLV J00587 http://www. Selsing E. lambda CACCTTCCTCTGAAGAGCTCGAGACTAACAAGGCCAC CTITDFYPGVVTVDWKVDGTPVTQGME AH005311 imgt.org/ et al. Proc 1 (CL) ACTGCTGTGTACGATCACTCATTTCTACCCAGGTGTG TTQPSKQSNNKYMASSYLTLTARAWER X58411 IMGT Natl. Acad GTGACAGTGGACTGGAAGGTACATCCTACCCCTGTC HSSYSCQVTHEGHTVEKSLSRADCS V00814 reportoire/ Sci USA. ACTCAGGGTATGGAGACAACCCAGCCTTGCAAACAG (SEQ ID NO: 38) index. 79, 4681- AGCAACAAGAAGTACATGGCTAGCAGCTACCTGACCC ghp?section 4685 (1982) TGACAGCAAGACCATGGGAAAGGCATAGCAGTTACA =locus PMID: GCTGCCAGGTCACTCATGAAGGTCACACTGTGGAGA Genes& 8812053 AGAGTTTGTGCCGTGCTGACTGTTCCTAG reportoire= Weiss S. (SEQ ID NO: 39) genetable& and W GE. species= EMBO J. mus musculus& 6, 927- group=IGKC 937 (1987) PMID: 3105891 Bernard O. et al. Cell, 15, 1133- 144 (1978) PMID: 103630 GGTCAGCCCAAGTCCACTCCCACTCTCACGGTGTTTC GQPKSTPTLTVTPPSSEELKENKATLV J00595 Selsing E. CACCTTCCTCTGAGGAGCTCAAGGAAAAGAAAGCCAC CLISNFSPSGVTVAWKANGTPITQGVD AH001968 et al. Proc ACTGGTGTGTCTGATTTCCAACTTTTCCCCGAGTGGT TSNPTKEDNKYMASSFLHLTSDQWRSH J00592 Natl. Acad GTGACAGTGGCCTGGAAGGCAAATGGTACACCTATCA NSFTCQVTHEGDTVEKSLSPAECL AH001967 Sci USA. CCCAGGGTGTGGACACTTCAAATCCCACCAAAGAGG (SEQ ID NO: 40) X58414 79, 4681- GCAACAAGTTCATGGCCACCAGCTTCCTACATTTGAC 4685 (1982) ATCGCACCAGTGGAGATCTCACAACAGTTTTACCTGT PMID: CAAGTTACACATGAAGCGGACAGTGTCGAGAAGAGT 8812053 CTGTCTCCTGGAGAATGTCTGTAA Wu G. et al, Cell 33, 77- 83 (1983) (SEQ ID NO: 41) PMID: 3109691 GGTCAGGCCAAGTGCACTCCCACACTGAGCATGTTTG GQPKSTPTLTMFPPSSEELQENKATLV J00585 Selsing E. CACCTTCCCCTGAGGAGCTCCAGGAAAACAAAGCCA CLISNFSPSGVTVAWKANGTPITQGVD AH005311 et al. Proc CACTCCTGTGTCTGATTTCCAATTTTTCCCCAAGTGG TSNPTKEDNKYMASSFLHLTSDQWRSH X58415 Natl. Acad TGTGACAGTGGCCTGGAAGGCAAATGGTACACCTATC NSFTCQVTHEGDTVEKSLSPAECL X58411 Sci USA. ACCCAGGGTGTGGACAGTTCAAATCCCACCAAAGAG (SEQ ID NO: 42) 79, 4681- GACAACAAGTACATGGGCAGCAGGTTCTTACATTTGA 4685 (1982) CATCGGAGCAGTGGAGATCTCACAACAGTTTTAGCTG PMID: CCAAGTTACACATGAAGGGGACACTGTGGAGAAGAG 8812053 TCTGTCTCCTGCAGAATGTCTGTAA Weiss S. (SEQ ID NO: 43) and W GE. EMBO J. 6, 927- 937 (1987) PMID: 3105891 Bovine Bovine IgG1 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC X62916 http://www. Symons D.B (Scientific Ig variant TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP imgt.org/ et a;. J. Name: Bos heavy 1 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGQTFT IMGT Immuno- taurus) chain GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGGGTGCA CNVAHPASSTKVDKAVDPTCKPSPCD reportoire/ genes, 14, con- CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTGTACT CCPPPELPGGPSVFIFPPKPKDTLTISG index. 273-283 stant CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD ghp?section (1987) region GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG VEVNTATTKPREEQFNSTYRVVSALRI =locus PMID: (CH1 CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA QHQDWTGGKEFKCKVHNEGLPAPIVRT Genes& 341517 CH3) AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGCTC ISRTKGPAREPQVYVLAPPQEELSKST reportoire= Symons D.B GCCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES genetable& et al, Mol. CAAGGACACCCTCAGAATCTCGGGAACGCCCGAGGTCA EDKYGTTPPQLDADSSYFLYSKLRVDR species= Immno., 26 CGTGTGTGGTGGTGGAGGTGGGCCACGATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS bovine taurus 341-350 GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA TSKSAGK* &group=IGKC (1989) CACAGCCACGACGAAGCCCAGAGAGGAGCAGTTCAACA (SEQ ID NO: 66) PMID: GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC 2513487 CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT Kacksovics CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACGA I. and TCTCCAGGACCAAAGGGCCGGGCCGCGAGCCGCAGGT Butler J.E. GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA Mol. GCACGGTCAGCCTCACCTGCATGGTCACCAGCTTCTAC Immunol, 33 CCAGACTACATCGCCGTGGAGTGGCAGAGAAAGGGGCA 189-195 GCCTGAGTCGGAGGACAAGTACGGCACCACCCCCGCC (1999) CAGCTGGACGCCGACAGCTCCTACTTCCTCTACAGCAA PMID: GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC 8849440 ACCTACACGTGTGTGGTGATGCACGAGGCCCTGCACAA Rabbani H. TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA et al. AATGA Immuno- (SEQ ID NO: 67) genetics, 46, 328- 331 (1997) PMID: 9218535 Saini S.S. et al Searol J. Immunol. 65, 32-8 (2007) PMID: 17212754 IgG1 GGCTCCAGGACAGGCGCGAAAGTCTAGCCTGTGAGTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC X16701 variant TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVVTVWNSGALKSGVHTFP (M25278) 2 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTCACC AVLQSSGLYSLSSMVTVPGSTSGQTFT GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGGGTGCA CNVAHPASSTKVDKAVDPTCKPSPCD CACCTTCCCCGCCGTCCTTCAGTCCTCCGGGCTGTACT CCPPPELPGCPSVFIFPPKPKDTLTISG CTCTCAGGAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD GGACAGAGCTTCACGTGCAACGTAGCCCACCCGGCCAG VEVNTATTKPREEQFNSTYRVVSALRI CAGCACCAAGGTGGACAAGGCTGTTGATCCCACATGCA QHQDWTGGKEFKCKVHNEGLPAPIVRT AACCATCACCCTGTGACTGTTGCCCACCCCCTGAGCTC ISRTKGPAREPQVYVLAPPQEELSKST CGCGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES CAAGCACACCCTCACAATCTCCGCAACGCCCGAGGTCA EDKYGTTPPQLDADSSYFLYSKLRVDR CGTGTGTGCTGCTGGACGTGGGCCACCATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGGTAAA TSKSAGK* CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA (SEQ ID NO: 68) GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCACCAC CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT CCACAACGAAGGCCTCCCGGCCCCCATCGTGAGGACCA TCTCCAGGACCAAAGGGCCGGCCCGGGAGCCCCAGGT GTATGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAAAA GCACCGTCAGCCTCACCTGCATGGTCACCACCTTCTAC CCAGACTACATCGCCGTGGACTGGCAGACAAACGGGCA GCCTGAGTCGGAGGACAAGTACGGCACGACCCCGGGC CAGCTGGACGCCGACAGCTCCTACTTCCTGTACAGCAA GCTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGAC ACCTACACGTCTGTGGTGATGCACGAGGCCCTCCACAA TCACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTA AATGA (SEQ ID NO: 69) IgG1 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC S82407 variant TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP 3 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGTQTF GTGACCTGGAACTCGGGTGCCCTGAACAGCGGCGTGCA TCNVAHPASSTKVDKAVDPRCKTTGD CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT CCPPPELPGGPSVFIFPPKPKDTLTISG CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA TPEVTCVVVDVGHDDPEVKFSWFVDD GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC VEVNTATTKPREEQFNSTYRVVSALRI CAGCAGCACCAAGGTGGACAAGGCTGTTGATCCCAGAT QHQDWTGGKEFKCKVHNEGLPAPIVRT GCAAAACAACCTGTGACTGTTCCCCACCGCCTGAGCTC ISRTKGPAREPQVYVLAPPQEELSKST CCTGGAGGACCCTCTGTCTTCATCTTCCCACCGAAACC VSLTCMVTSFYPDYIAVEWQRNGQPES CAAGGACACGCTCACAATCTCGGGAACGCCCGAGGTCA EDKYGTTPPQLDADGSYFLYSRLRVDR CGTGTGTGGTGGTGGACGTGGGCCACGATGACCCCGAG NSWQEGDTYTCVVMHEALHNHYTQKS GTGAAGTTCTCCTGGTTCGTGGACGACGTGGAGCTAAA TSKSAGK* CACAGCCACGACGAAGCCGAGAGAGGAGCAGTTCAACA (SEQ ID NO: 70) GCACCTACCGCGTGGTCAGCGCCCTGCGCATCCAGCAC CAGGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAGGT CCACAACGAAGGCCTCCCAGCCCCCATCGTGAGGACCA TCTCCACGACCAAAGGGCCGGCCCGGGACGCGGAGGT GTATGTGCTGGCCCCAGCCCAGGAAGAGGTCAGCAAAA GCACGGTCAGCCTCACGTGCATGGTCACCAGCTTCTAC CCAGACTACATCGCCGTGGAGTGGCAGAGAAATGCGCA GCCTGAGTCAGAGGACAAGTACGGCACGACCCCTCCCC AGCTGGACGCCGACCGCTCCTACTTCCTGTACACCAGG CTCAGGGTGGACAGGAACAGCTGGCAGGAAGGAGACA CCTACACGTGTGTGGTGATGGACGAGGCCCTGCACAAT CACTACACGCAGAAGTCCACCTCTAAGTCTGCGGGTAA ATGA (SEQ ID NO: 71) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC ASTTAPKVYPLASSCGDTSSSTVTLGC S82407 variant CAGCTGCGCAGACACATCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP 1 GCTGCCTGGTGTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPASSSGQTFT GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA CNVAHPASSTKVDKAVGVSIDCSKCHN CACCTTCCCGGCTGTCCTTCAGTCCTCCGGGCTCTACT QPCVREPSVFIFPPKPKDTLMITGTPEV CTCTCAGCAGCATGGTGACCGTGCCCGCCAGCAGCTCA TCVVVNVGHDNPEVQFSWFVDDVEVH GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG TARSKPREEQFNSTYRVVSALPIQHQD CAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCATTG WTGGKEFKCKVNNKGLSAPIVRIISRSK ACTGCTCCAAGTGTCATAACCAGCCTTGCGTGAGGGAA GPAREPQVYVLDPPKEELSKSTLSVTC CCATCTGTCTTCATCTTCCCACCGAAACCCAAAGACAC MVTGFYPEDVAVEWQRNRQTESEDKY CCTGATGATCACAGGAACCCCCCACCTCACGTGTGTCG RTTPPQLDTDRSYFLYSKLRVDRNSWQ TGGTGAACGTGGGCCACGATAACCCCGAGGTGCAGTTC EGDAYTCVVMHEALHNHYMQKSTSKS TCCTGGTTCGTGGATGACGTGGAGGTGCACACGGCCAG AGK* GTCGAAGCCAAGAGAGGAGCAGTTCAACAGCACGTACC (SEQ ID NO: 72) GCGTGGTCACCGCCCTGCGCATCCAGCACCAGGACTGG ACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAACAA AGGCCTCTCGGCCCCCATCGTGAGGATCATCTCCAGGA GCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGTCCT GGACCCACCCAAGGAAGAGCTCAGCAAAAGCACGCTCA GCGTCACCTGCATGGTCACCCGCTTCTACCCAGAAGAT GTAGCCGTGGAGTGGCAGAGAAACCGGCAGACTGAGTC GGAGGACAAGTACCGCACGACCCCGCCCCAGCTGGAC ACCGACCGCTCCTACTTCCTGTACAGCAAGCTCAGGGT GGACAGGAACAGCTGCCACGAAGGAGACGCCTACACG TGTGTGGTGATGCACGAGGCCCTGCACAATCACTACAT GCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA (SEQ ID NO: 73) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGACTTC ASTTAPKVYPLSSCCGDKSSSTVTLGC M36946 variant TTGCTGCGGGGACAAGTCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP (X08703) 2 GCTGCCTGGTGTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPGSTSGQTFT GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA CNVAHPASSTKVDKAVGVSSDCSKPN CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT NQHCVREPSVFIFPPKPKDTLMITGTPE CTCTCAGCAGCATGGTGACCGTGCCCGGCAGCACCTCA VTCVVVNVGHDNPEVQFSWFVDDVEV GGACAGACCTTCACCTGCAACGTAGCCCACCCGGCCAG HTARTKPREEQFNSTYRVVSALPIQHQ CAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCAGTG DWTGGKEFKCKVNIKGLSASIVRIISRS ACTGGTCCAAGCCTAATAACCAGCATTGCGTGACGGAA KGPAREPQVYVLDPPKEELSKSTVSVTC CCATCTGTCTTCATCTTCCCACCGAAACCCAAAGACAC MVIGFYPEDVDVEWQRDRQTESEDKYR CCTGATGATCACAGGAACCCCCGAGGTCACGTGTGTGG TTPPQLDADRSYFLYSKLRVDRNSWQR TGGTGAACGTGGGCCACGATAACCCCGAGGTGCAGTTC GDTYTCVVMHEALHNHYMQKSTSKSA TCCTGGTTCGTGGACGACGTGGAGGTGCACACGGCCAG GK* GACGAAGCCGAGAGAGGACCAGTTCAACAGCACGTACC (SEQ ID NO: 74) GCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGACTGG ACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACATCAA AGGCCTCTCGGCCTCCATCGTGAGGATCATCTCCAGGA GCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGTCCT GCACCCACCCAAGGAACAGCTCAGCAAAAGCACGGTCA GCGTCACCTGCATGGTCATCGGCTTCTACCCAGAAGAT GTAGACGTGGAGTGGCAGAGAGACCGGCAGACTGAGTC GGAGGACAAGTACCGCACGACCCCGCCCCAGCTGGAC GCCGACCGCTGGTACTTCCTGTACAGCAAGCTCAGGGT GGACAGGAACAGCTGGCAGAGAGGAGACACCTACACGT GTGTGGTGATGCACGAGGCCCTGCACAATCACTACATG CAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA (SEQ ID NO: 75) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGAGTTC ASTTAPKVYPLSSCCGDKSSSGVTLGC X16702 variant TTGCTGCGGGGACAAGTCCAGCTCGGGGGTGACCCTGG LVSSYMPFPVTVTWNSGALKSGVHTFP (M25278) 3 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVLQSSGLYSLSSMVTVPASSSGTQTF GTGACCTGGAAGTCGGGTGCCCTGAAGAGCGGCGTGCA TCNVAHPASSTKVDKAVGVSSDCSKP CACCTTCCCGGCCGTCCTTCAGTCCTCCGGGCTCTACT NNQHCVREPSVFIFPPKPKDTLMITGTP CTCTCAGCACCATCGTCACCGTGCCCGCCAGCAGCTCA EVTCVVVNVGHDNPEVQFSWFVDDVE GGAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC VHTARTKPREEQFNSTYRVVSALPIQH CAGCAGCACCAAGGTGGACAAGGCTGTTGGGGTCTCCA QDWTGGKEFKCKVNIKGLSASIVRIISR GTGACTGCTCCAAGCCTAATAACCAGCATTGCGTGAGG SKGPAREPQVYVLDPPKEELSKSTVSLT GAACCATCTGTCTTCATCTTCCCACCGAAACCCAAAGA CMVIGFYPEDVDVEWQRDRQTESEDKY CACCCTGATGATCACAGGAACGCCGGAGGTCACGTGTG RTTPPQLDADRSYFLYSKLRVDRNSWQ TGCTGCTGAACGTGGGCCACGATAACCCCGAGGTGCAG RGDTYTCVVMHEALHNHYMQKSTSKS TTCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC AGK* CAGGACGAAGCCGAGAGAGGAGCAGTTCAACAGCACGT (SEQ ID NO: 76) ACCGCGTGGTCAGCGCCCTGCCCATCCAGCACCAGGAC TGGACTGGAGGAAAGGAGTTCAAGTGCAAGGTCAACAT CAAAGGCCTCTCGGCCTCCATCGTGAGGATCATCTCCA GGAGCAAAGGGCCGGCCCGGGAGCCGCAGGTGTATGT CCTGGACCCACCCAAGGAAGAGCTCAGCAAAAGCACGG TCAGCCTCACCTGGATGGTCATCGGCTTCTACCCAGAA GATGTAGACGTGGAGTGGCAGAGAGACCGGCAGACTGA GTCGGAGGACAAGTACCGCACGACCCCCCCCCAGCTG GACGCCGAGGGCTCCTACTTCCTGTACAGCAAGCTGAG GGTGGACAGGAACAGCTGCCAGAGAGGAGACACCTAC ACGTGTGTGGTGATGCAGGAGGCCCTGCACAATCACTA CATGCAGAAGTCCACCTCTAAGTCTGCGGGTAAATGA (SEQ ID NO: 77) U63638 IgG3 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC AKTTAPKVYPLASSCGDTSSSTVTLGC variant CAGCTGCGGAGACACATGCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTWNSGALKSGVHTFP 1 GCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTGACC AVRQSSGLYSLSSMVTVPASSSETQTF GTGACCTGGAACTCGGGTGCCCTGAAGAGCGGCGTGCA TCNVAHPASSTKVKDAVTARRPVPTTP CACCTTCCCGGCCGTCCGGCAGTGCTCTGGGCTGTACT KTTIPPGKPTTPKSEVEKTPCQCSKCP CTCTCAGCAGCATGGTCACTGTGCGGGCCAGCAGCTCA EPLGGLSVFIFPPKPKDTLTISGTPEVT GAAACCCAGACCTTCACCTGCAACGTAGCCCACCCGGC CVVVDVGQDDPEVQFSWFVDDEVHT CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC ARTKPREEQFNSTYRVVSALRIQHQDW GTCCAGTCCCGACGACGCCAAAGAGAACTATCCCTCCT LQGKEKCKVNNKGLPAPIVRTISRTKG GGAAAACCGACAACCCCAAAGTGTGAAGTTGAAAAGAC QAREPQVYVLAPPREELSKSTLSLTCLI ACCCTGCCAGTGTTCCAAATGCCCAGAACCTCTGGGAG TGFYPEEIDVEWQRNGQPESEDKYHTT GACTGTCTGTCTTCATCTTCCCACCGAAACCCAAGCAC APQLDADGSYFLYSKLRVNKSSWQEG ACCCTCACAATCTCGGGAACGCCCGAGGTCACGTGTGT DHYTCAVMHEALRNHYKEKSISRSPGK GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG * TTCTCCTGGTTGGTGGACGACGTGGAGGTGCACACGGC (SEQ ID NO: 78) CAGGACGAAGCCGACAGAGGACCAGTTCAACAGCACCT ACCGCGTGGTCAGCGCCCTGCGCATCCAGCACCAGGA CTGGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA ACAAAGGCCTCCCGGCCCCCATTGTGAGGACCATCTCC AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG TCCTGGCCCCACCCCGGGAAGAGCTCAGGAAAAGCACG CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTG AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG GATGCTGACGGCTCCTACTTCCTGTACAGCAAGCTCAC GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA CGTGTGCAGTGATGCACGAAGCTTTACGGAATCACTAC AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA (SEQ ID NO: 79) IgG3 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCATC ASTTAPKVYPLASRCGDTSSSTVTLGC U63639 variant CCGCTGCGGAGACACATCCAGCTCCACCGTGACCCTGG LVSSYMPEPVTVTVWNSGALKSGVHTFP 2 GCTGCCTGGTCTCCAGCTACATCCCCGACCCCGTGACC AVLQSSGLYSLSSMVTVPASTSETQTF GTGACCTGGAACTCGGGTGCCCTGAAGACTCCCGTGCA TCNVAHPASSTKVDKAVTARRPVPTTP CAGCTTCCCGGCCGTCGTTGAGTCCTCCGGGCTGTACT KTTIPPGKPTTQESEVEKTPCQCSKGP CTCTCAGCAGGATGGTGACCGTGCCCGCCAGCACCTCA EPLGGLSVFIFPPKPKDTLTISGTPEVT GAAACCCAGACCTTCACCTGCAAGGTAGCCCACCCGGC CVVVDVGQDDPEVQFSWFVDDVEVHT CAGCAGCACCAAGGTGGACAAGGCTGTCACTGCAAGGC ARTKPREEQFNSTYRVVSALRIQHQDW GTCCAGTCCCGACGACGCCAAAGAGAACCATCCCGCCT LQKKEFKCKVNNKGLPAPIVRTISRTKG GGAAAACCCACAACCCAGGAGTCTGAAGTTGAAAAGAC QAREPQVYVLAPPREELSKSTLSLTCLI ACCCTGCCAGTGTTGCAAATGCCCAGAACCTCTGGGAG TGFYPEEIDVEWQRNGQPESEDKYHTT GACTGTCTGTCTTCATCTTCCCACCGAAACCCAAGGAC APQLDADGSYFLYSRLRVNKSSWQEG ACCCTCACAATCTCGGGAACGCCCGAGGTCACGTGTGT DHYTCAVMHEALRNHYKEKSISRSPGK GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGCAG * TGCTCCTGGTTCGTGGACGACGTGGAGGTGCACACGGC (SEQ ID NO 80) CAGGACGAAGCCGAGAGAGGAGCAGTTCAAGAGCAGCT ACCCCGTGGTCAGGGCCCTGCGCATCCAGCACCAGGA CTCGCTGCAGGGAAAGGAGTTCAAGTGCAAGGTCAACA ACAAAGGCCTCCCGGCCCCCATTGTGAGGAGCATCTCC AGGACCAAAGGGCAGGCCCGGGAGCCGCAGGTGTATG TCCTGGCCCCACCCCGGGAAGAGCTCAGCAAAAGCACG CTCAGCCTCACCTGCCTGATCACCGGTTTCTACCCAGA AGAGATAGACGTGGAGTGGCAGAGAAATGGGCAGCCTC AGTCGGAGGACAAGTACCACACGACCGCACCCCAGCTG GATGCTGACGGCTGCTACTTCCTGTACAGCAGGCTCAG GGTGAACAAGAGCAGCTGGCAGGAAGGAGACCACTACA CGTGTGCAGTGATGCATGAAGCTTTACGGAATCACTAC AAAGAGAAGTCCATCTCGAGGTCTCCGGGTAAATGA (SEQ ID NO: 81) Bovine Ig CAGCCCAAGTCCCCACCCTCGGTCACCCTGTTCCCGCC QPKSPPSVTLFPPSTEELNGNKATLVC X62917 Not Chen L. et Ig lambda CTCCAGGGAGGAGCTCAACGGCAACAAGGCCACCCTG LISDFYPGSVTVVWKADGSTITRNVETT registerd al., Vet. light GTGTGTCTCATCAGCGACTTCTACCCGGGTAGCGTCTAC RASKQSNSKYAASSYLSLTSSDWKSKG Immunol. chain CGTGGTCTCGAAGGCAGACGGCACCACCATCACCCGCA SYSCEVTHEGSTVTKTVKPSECS* Immuno- con- ACGTGGAGACCACCCGGGCCTCCAAACAGAGCAACAG (SEQ ID NO: 64) pathol, stant CAAGTAGGCGGCCAGCAGCTACCTGAGGCTGACGAGCA 124, 284- region GCGACTGGAAATCGAAAGGCAGTTACAGCTGCGAGGTC 294 (2008) (CL) ACGCACGAGGGGAGCACCGTGACGAAGACAGTGAAGC PMID: CCTCAGAGTGTTCTTAG 18538861 (SEQ ID NO: 65) Canine Canine IgG-D GCCTGCACCACGGCCGCCTCGGTTTTCCCACTGGCC ASTTAPSVFPLAPSCGSTSGSTVALAC AF354267 http://www. Tang L. (Specific Ig CCCAGCTGCGGGTCCACTTCCGGCTCCACGGTGGCC LVSGYFPEPVTVSWNSGSLTSGVHTFP imgt.org/ et al., Name: heavy CTGGCCTGCCTGGTGTCAGGCTACTTCCCCGAGCCT SVLQSSGLYSLSSTVTVPSSRWPSETF IMGT Vet Immuno- Canis chain GTAACTGTGTCCTGGAATTCCGGCTCCTTGACCAGC TCNVVHPASNTKVDKPVPKESTCKCIS reportoire/ pathol 80 Lupus con- GGTGTGCACACCTTCCCGTCCGTCGTGCAGTCCTCA PCPVPESLGGPSVFIFPPKPKDILRITR index. (3-4), famililaris) stant GGGCTCTACTCCCTCAGCAGGACGGTGACAGTGCCC TPEITCVVLDLGREDPEVQISWFVDGKE ghp?section 259-270 region TCCAGCAGGTGGCCCAGCGAGACCTTCACCTGCAAC VHTAKTQPREQQFNSTYRVVSVLPIEHQ =locus (2001) (CH1 GTGGTCCACCCGGCCAGCAACACTAAAGTAGACAAG DWLTGKEFKCRVNHIGLPSPIERTISKA Genes& PMID: CH3) CCAGTGCCCAAAGAGTCCACCTGCAAGTGTATATCC RGQAHQPSVYVLPPSPKELSSSDTVTL reportoire= 11457479 CCATGCCCAGTCCCTGAATCACTGGGAGGGGCTTCG TCLIKDFFPPEIDVEWQSNGQPEPESKY genetable& GTCTTCATCTTTCCCCCGAAACCCAAGGACATCCTC HTTAPQLDEDGSYFLYSKLSVDKSRWQI species= AGGATTACCCGAACACCCGAGATCACCTGTGTGGTG QGDTFTCAVMHEALGNHYTDLSLSHS dog TTAGATGTGGGCGGTGAGGAGCCTGAGGTGCAGATC PGK* &group=IGHC AGCTGGTTCGTGGATGGTAAGGAGGTGCACACAGCC (SEQ ID NO 84) AAGACGCAGCCTCGTGAGCAGCAGTTCAACAGCACC TACCGTGTGGTCAGCGTCCTCCCCATTGAGCACCAG GACTGGCTCACCGGAAAGGAGTTCAAGTGCAGAGTC AACCACATAGGCCTCCCGTCCCCCATCGAGAGGACT ATCTCCAAAGCCAGAGGGCAAGCCCATCAGCCCAGT GTGTATGTCCTGCCACCATCCCCAAAGGAGTTGTCA TCCAGTGACACGGTCACCCTGACCTGCCTGATCAAA GACTTCTTCCCACCTGACATTGATGTGGAGTGGCAG AGCAATGGACAGCCGGAGCCCGAGAGCAAGTACCAC ACGACTGCGCCCCAGCTGGACGAGGACGGGTCCTAC TTCCTGTACAGCAATTCTCTCTGTGGACAAGAGCCGC TGGCAGCAGGGAGACACCTTCACATGTGCGGTGATG CATGAAGCTCTACAGAACCACTACACAGATCTATCCC TCTCCCATTCTCCGGGTAAATGA (SEQ ID NO: 85) Canine Ig CAGCCCAAGGCGTCCCCCTCGGTCACACTCTTCCCG QPKASPSVTLFPPSSEELGANKATLVC E02824 not None Ig lambda CCCTCCTCTGACGAGCTCGGCCCCAACAAGGCCACC LISDFYPSGVTVAWKASGSPVTQGVET registered light (CL) CTGGTGTGCCTCATCAGCGACTTCTACCCCAGCGGC TKPSKQSNNKYAASSYLSLTPDKWKSHI chain GTGACGGTGGCCGGAAGGCAAGCGGCAGCCCCGT SSFSCLVTHEGSTVEKKVAPAECS* con- CACCCAGGGCGTGGAGACGACCAAGCCCTCCAAGCA (SEQ ID NO: 82) stant GAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAG region CCTGACGCCTGACAAGTGGAAATCTCACAGCAGCTT CAGCTGCCTGGTCACGCACGAGGGGAGCACCGTGG AGAAGAAGGTGGCCCCCGCAGAGTGCTCTTAG (SEQ ID NO: 83) Ovian Ovine IgG1 GCCTCAACAACACCCCCGAAAGTGTACCCTCTGACT ASTTPPKVYPLTSCCGDTSSSIVTLGC X69797 http://www. Dufour V. (Scientific Ig TCTTGCTGCGGGGACACGTCCAGCTCCATCGTGACC LVSSYMPEPVTVTWNSGALTSGVHTF imgt.org/ et al. J. Name: chain CTGGGCTGCCTGGTCTGCAGCTATATGCCCGAGCCG PAILQSSGLYSLSSVVTVPASTSGAQT IMGT Immunol, Ovis light GTGACCGTGACCTGGAACTCTGGTGCCCTGACCAGG FICNVAHPASSTKVDKRVEPGCPDPCK reportoire/ 156, 2163- aries) con- GGCGTGCACACCTTCCCGGCCATCCTGCAGTCCTCC HCRCPPPELPGGPSVFIFPPKPKDTLTI index. 2170 (1996) stant GGGCTCTACTCTCTCAGCAGCGTGGTGACCGTGCCG SGTPEVTCVVVDVGQDDPEVQFSWFV ghp?section PMID: region GCCAGCACCTCAGGAGCCCAGACCTTCATCTGCAAC DNVEVRTARTKPREEQFNSTFRVVSAL =locus 8690905 CH1 GTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG PIQHQDWTGGKEFKCKVHNEALPAPIV Genes& CH3) CGTGTTGAGCCCGGATGCCCGGACCCATGCAAACAT RTISRTKGQAREPQVYVLAPPQEELSK reportoire= TGCCGATGCCCACCCCCTGAGCTCCCCGGAGGACC STLSVTCLVTGFYPDYIAVEWQKNGQP genetable& GTCTGTCTTCATCTTCCCACCGAAACCCAAGGACAC ESEDKYGTTTSQLDADGSYFLYSRLRV species= CCTTACAATCTCTGGAACGCCCGAGGTCACGTGTGT DKNSWQEGDTYACVVMHEALHNHYTQ sheep GGTGGTGGACGTGGGCCAGGATGACCCCGAGGTGC KSISKPPGK* &group=IGHC ACTTCTCCTGGTTCGTGGACAACGTGGACGTGCCCA (SEQ ID NO: 90) CGGCCAGGACAAAGCCGAGAGAGGAGCAGTTCAACA GCACCTTCCGCGTGGTCAGCGCCGTGCCCATCCAGC ACCAAGACTGGACTGGAGGAAAGGAGTTCAAGTGCA AGGTCCACAACGAAGCCCTCCCGGCCCCCATCGTGA GGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAG CCGGAGGTGTACGTCCTGGCCCCACCCCAGGAAGAG CTCAGCAAAAGCACGCTCAGCGTCACCTGCCTGGTC ACCGGCTTCTACCCAGACTACATCGCCGTGGAGTGG CAGAAAAATGGGCAGCCTGAGTCGGAGGACAAGTAC GGCACGACCACATCCCAGCTGGACGCCGACGGCTCC TACTTGCTGTACAGCAGGCTCAGGGTGGAGAAGAAC AGCTGGCAAGAAGGAGACACCTACGCGTGTGTGGTG ATGCACGAGGCTCTGCACAACCACTACACACAGAAG TCGATCTCTAAGCCTCCGGGTAAATGA (SEQ ID NO: 91) IgG2 GCCTCCACCACAGCCCCGAAAGTCTACCCTCTCACT ASTTAPKVYPLTSCCGDTSSSSSIVTL X70983 Clarkson TCTTGCTGCGGGGACACGTCCAGCTCCAGCTCCATC GCLVSSYMPEPVTVTWNSGALTSGVH C.A. et GTGACCCTGGGCTGCCTGGTCTCCAGCTATATGCCC TFPAILQSSGLYSLSSVVTVPASTSGA al, Mol. GAGCCGGTGACCGTGACCTGGAACTCTGGTGCCCTG QTFICNVAHPASSAKVDKRVGISSDYS Immunol, ACCAGCGGCGTGCACACCTTCCCGGCCATCCTGCAG KCSKPPCVSRPSVFIFPPKPKDSLMITG 20 1195- TCCTCCGGGCTCTACTCTCTCAGCAGCGTGGTGACC TPEVTCVVVDVGQGDPEVGFSWFVDN 1204 (1993) GTGCCGGCCAGCAGCTCAGGAGCCCAGACCTTCATC VEVRTARTKPREEQFNSTFRVVSALPI PMID: TGCAACGTACCCCACCCCCCCAGCACCGCGAACGTG QHDHWTGGKEFKCKVHSKGLPAIVRT 8413324 GACAAGCGTGTTGGGATCTCCAGTGACTACTCCAAG ISRAKGQAREPQVYVLAPPQEELSKST TGTTCTAAACCGCCTTGCGTGAGCCGACCGTCTGTC LSVTCLVTGFYPDYIAVEWQRARQPES TTCATCTTCCCCCCGAAACCCAAGGACAGCCTCATG EDKYGTTTSQLDADGSYFLYSRLRVGK ATCACAGGAACGCCCGAGGTCACGTGTGTCGTGGTG SSWQRGDTYACVVMHEALHNHYTGKS GACGTGGGCCAGGGTGACCCCGAGGTGCAGTTCTCC ISKPPGK* TGGTTCGTGGACAACGTGGAGGTGCGCACGGCCAGG (SEQ ID NO: 92) ACAAAGCCGAGAGAGGAGCAGTTCAACAGCACCTTC CGCGTGGTCAGCGCCCTGCCCATCCAGCACGACCAC TGGACTCGAGGAAAGGAGTTCAAGTGCAAGGTCCAC AGCAAAGCCCTCCCGGCCCCCATCGTGAGGACCATC TCCAGGGCCAAAGGGCAGGCCCGGGAGCCGCAGGT GTAGGTCCTGGCCCCACCCCAGGAAGAGCTCAGCAA AAGCACGCTCAGCGTCACCTGCCTGGTCACCGGCTT CTACCCAGACTACATCGCCGTGGAGTGGCAGAGAGC GCGGCAGCCTCAGTCCGAGGAGAAGTACGCCACGAC CACATCCCAGCTGGACGCCGACGGCTCCTACTTCCT GTACAGCAGGCTCAGGGTGGACAAGAGCAGCTGGCA AAGAGGAGACACCTAGGCGTGTGTGGTGATGCACGA GGCTCTGCACAACCACTACACACAGAAGTCGATCTC TAAGCCTCCGGGTAAATGA (SEQ ID NO: 93) Ovine Ig CCATCCGTCTTCCTCTTCAAACCATCTGAGGAACAG PSVFLFKPSEEQLRTGTVSVVCLVNDF X54110 Not Jenne C.N. Ig kappa CTGAGGACCGGAACTGTCTCTGTCGTCTGCTTCGTG YPKDINVKVKVDGVTQNSNFQNSFTDQ registered et al. Dev light (CL) AATGATTTCTACCCCAAAGATATCAATGTCAAGGTGA DSKKSTYSLSSTLTLSSSEYQSHNAYA Comp. chain AAGTGGATGGGGTTACCCAGAACAGCAACTTCCAGA CEVSHKSLPTALVKSFNKNEC* Immunol. con- ACAGCTTCACAGACCAGGACAGRAAGAAAAGCACCT (SEQ ID NO: 86) 30 (1-2), stant ACAGCCTCAGCAGCACCCTGAGACTGTCCAGCTCAG 165-174 region AGTACCAGAGCCATAACGCCTATGCGTGTGAGGTCA (2006) GCCACAAGAGCCTGCCCACCGCCCTCGTCAAGAGCT PMID: TCAATAAGAATGAATGTTAG 16083958 (SEQ ID NO: 87) Ig GGTCAGCCCAACTCCGCACCCTCGGTCACCCTGTTC GQPKSAPSVTLFPPSTEELSTNKATVV AY734681 lambda CCGCCTTCCACGGAGGAGCTCAGTAGCAAGAAGGCC CLINDFYPGSVNVVWKADGSTINQNVK (CL) ACCGTGGTGTGTCTCATCAACGACTTCTACCCGGGT TTQASKQSNSKYAASSYLTLTGSEWKS AGCGTGAACGTGGTCTGGAAGGCAGATGGCAGCACC KSSYTCEVTHEGSTVTKTVKPSECS* ATCAATCAGAACGTGAAGACCACCCAGGCCTCCAAA (SEQ ID NO: 88) CAGAGCAACAGCAAGTACGCGGCCAGCAGCTACCTG ACCCTGACGGGCAGCGAGTGGAAGTCTAAGAGCAGT TAGACCTGCGAGGTCACGCACGAGGGGAGCACCGTG ACGAAGACAGTGAAGCCCTCAGAGTGTTCTTAG (SEQ ID NO: 89) Porcine Por- IgG3 GCCCCCAAGACGGCCCCATCGGTCTACCCTCTGGCCCCCTGCGGCAC APKTAPSVYPLAPCGRDTSGPNVALG U03781 http://www. Butler J.E. (Scientific cine CGACACGTCTC CLASSYFPEPVTMTWNSGALTSGVHT imgt.org/ et al. Name: Sus Ig GCCCTAACGTGGCCTTGGGGTGCCTGGCCTCAAGCTACTTCCCCGAG FPSVLQPSGLYSLSSMVTVPASSLSS IMGT Immuno- serpa) heavy CCAGTGACCATG KSYTCNVNHPATTTKVDKRVGTKTKP reportoire/ genetics chain ACCTGGAACTGGGGCGCCCTGACCAGTGGCGTGCATACCTTCCCATC PCPICPGCEVAGPSVFIFPPKPKDTLMI index. 61(3) con- CGTCCTCCAGCC SQTPEVTCVVVDVSKEHAEVQFSWYV ghp?section 209-230 stant CTCAGGGCTCTACTCCCTCAGCAGCATGGTCACCGTGCCCGCCAGCA DGVEVHTAETRPKEEQFNSTYRVVSV =locus PMID: region GCCTGTCGAGCA LPIQHQDWLKGKEFKCKVNNVDLPAP Genes& 19028748 (CH1 AGAGCTACACCTGCAATGTCACCACCCGGCCACCACCACCAAGGTGG TRTISKAIGQSREPQVYTLPPPAEELS reportoire= Kaeskoves CH3) ACAAGCGTGTT RSKVTVTCLVIGIFYPPDIHVEWKSNGQ genetable& I et al. GGAACAAAGACCAAACCACCATGTCCCATATGCCCAGGCTGTGAAGT PEPEGNYRTTPPQDDVDGTFFLYSKL species= J. Immunol GCCCGGGCCCTC AVDKARWDHGETFECAVMHEALHNHY sheep 153(8) GGTCTTCATCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCC TQKSISKTGGK* &group=IGHC 3585-3573 AGACCCCCGAGG (SEQ ID NO: 94) (1994) TCACGTGCGTGGTGGTGGACGTCAGCAAGGAGCACGCCGAGCTCCAG PMID: TTCTCCTGGTAC 7930579 GTGGACGGCGTAGAGGTGCACACGCCCGACACGAGACCAAAGGAGGA GCAGTTCAACAG CACCTACCGTGTCCTCAGCGTCCTGCCCATCCAGCACCACGACTGGC TGAAGGGGAAGG AGTTCAAGTGCAAGGTCAACAACGTAGACCTCCCAGCCCCCATCACG AGGACCATCTCC AAGGCTATAGGGCAGAGCCGGGAGCGGCAGGTGTACACCCTGCCCCC ACCCGCCGAGG ACCTGTCCAGGAGCAAAGTCACCGTAACCTGCCTGGTCATTGGCTTC TACCCACCTGAC ATCCATGTTGAGTGGAAGAGCAACGGACAGCCGGAGCCAGAGGGCAA TTACCGCACCAC CCCGCCCCAGCAGGACGTGGACGGGACCTTCTTCCTGTACAGCAAGC TCGCGGTGGACA AGGCAAGATGGGACCATGGAGAAACATTTGAGTGTGCCGTGATGCAC GAGGCTCTGGAC AACCACTACAGCCAGAAGTCCATCTCCAAGACTCAGGGTAAATGA (SEQ ID NO: 95) IgG3 GCCCCCAAGACGGCCCCATCGGTCTACCCTCTGGCCCCCTGCCGCAG APKTAPSVYPLAPCGRDVSGPNVALG U003718 GGACGTCTCTG CLASSYFPEPVTVTWNSGALTSGVHT GCCCTAACGTGGCCTTGGGCTGCCTGGCCTCAAGCTACTTCCCCGAG FPSVLQPSGLYSLSSMVTVPASSLSS CCAGTGACCGTG KSYTCNVNHPATTTKVDKRVGIHQPQ ACCTGGAACTCGGGCGCCCTGACCACTGGCGTGCACACCTTCCCATC TCPICPGCEVAGPSVFIFPPKPKDTLMI CGTCCTGCAGCC SQTPEVTCVVVDVSKEHAEVQFSWYV GTCAGGGCTCTACTCCCTCACCAGCATGCTCACCGTGCCGGCCAGCA DGVEVHTAETRPKEEQFNSTYRVVSV GCCTGTCCAGCA LPIQHQDWLKGKEFKCKVNNVKLPAPI AGAGCTACACCTGCAATGTCAACCACCCGGCCAGCACCACCAAGGTG TRTISKAIGQSREPQVYTLPPPAEELS GACAAGCGTGTT RSKVTLTCLVIGFYPPDIHVEWKSNGQ GGAATACACCAGCCGCAAACATGTCCCATATGCCCAGGCTGTGAAGT PEPENTYRTTPPQQDVDGTFLYSKL GGCCGGGCCCTC AVDKARWDHGDKFECAVMHEALHNH GGTCTTCATCTTCCCTCCAAAACCCAACGACACCCTCATGATCTCCC YTQKSISKTQGK* AGACCCCCGAGG (SEQ ID NO: 96) TCACGTGCGTGGTGGTGGACGTCAGCAAGGAGCACGCCGAGGTCCAG TTCTCCTGGTAC GTGGACGGCGTAGAGGTGCACACGGCCGAGACGAGACCAAAGGAGGA GCAGTTCAACAG CACCTACCGTGTGGTCAGCGTCGTGCCCATCCAGCACGAGGACTGGC TGAAGGGGAACG AGTTCAAGTGCAAGGTCAACAACGTAGACCTCCCACCCCCCATCACC AGGACCATCTCC AAGGCTATAGGGCAGAGCCGGGAGCCGCAGGTGTACACCCTGCCCCC ACCCGCCGAGG AGCTGTCCAGGAGCAAAGTCACGCTAAGCGCCTCCCCATTGCCTTCT ACCCACCTGAC ATCCATGTTGAGTGGAAGAGCAACGGACAGCCGGAGCCAGAGAACAC ATACCGCACCAC CCCGCCCCAGCAGGACGTGGACGGGACCTTCTTCCTGTACAGCAAAC TCGCGGTGGACA AGGCAACATCGGAOCATGGAGACAAATTTGAGTGTCCGCGGAGGGAC CACGCGCTGCAC AACCACTACACCCAGAAGACCATCTCCAAGACTCAGGGTAAATGA (SEQ ID NO: 97) IgG2 GCCCCCCAAGACGCCCCCATCGGTCTACCCTCTGGCCCCCTGCAGCA APKTAPSVYPLAPCSRDTSGPNVALG U03779 GGGACACGTCTG CLASSYFPEPVTVTWNSGALSSGVHT GCCCTAACGTGGCGTTGGGCTGCCTGGCCTCAAGCTACTTCCCCGAG FPSVLQPSGLYSLSSMVTVPASSLSS CCAGTGACCGTG KSYTCNVNHPATTTKVDKRVGTKTKP ACCTGGAACTCGGGCGCCCTGTCCAGTGGCGTGCATACCTTCCCATC PCPICPACESPGPSVFIFPPKPKDTLMI CGTCCTGCAGCC SRTPQVTCVVVDVSQENPEVQFSWYV GTCAGGGCTCTACTCCCTCAGCAGCATGGTGACCGTGCCGGCCAGCA GDVEVHTAQTRPKEEQFNSTYRVVSV GCCTGTCCAGCA LIIQHQDWLNGKEFKCKVNNKDLPAPI AGAGCTACACCTGCAATGTCAACCACCCGGCCACCACCACCAAGGTG TRISKAKGQTREPQVYTLPPHAEELS GACAAGCGTGTT RSKVSITCLVIGFYPPDIDVEWQRNGQ TGGAACAAAGACCAAACCACCATGTCCCATATGCCCAGCCTGTGAAT PEPGNYRTTPPQQDVDGTYFLYSKF CACCAGGGCCCTC SVDKASWQGGGIFQCAVMHEALHNHY TGGTCTTCATCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCC TQKSISKTPGK* CCGACACCCCAGG (SEQ ID NO: 98) TCACGTGCGTGGTGGTTGATGTGAGCCAGGAGAACCGGCAGGTCCAG TTCTGCTGGTAC GTGGACGGCGTAGAGGTGCACACGGCCCAGACGAGGCCAAAGGAGGA GCAGTTCAACAG CACCTACCGCGTGGTCAGCGTCCTACCCATCCAGCACCAGGACTCGC TGAACGGGAAGG AGTTCAAGTCCAAGGTCAACAACAAAGACCTCCCAGCCCCCATCACA AGGATCATCTCC AAGGCCAAAGGGCAGACCCGGGAGCCGCAGGTGTACACCCTGCCCCC ACACGCCGAGG TACCTCTCCACGAGCAAAGTCACCATAACCTGCCTGGTCATTCGCTT GTACCCACCTCAC ATCGATGTCGAGTGCCAAAGAAACGGACAGCCGGAGCCAGAGGGCAA TTACCGCACCAC CCCGCCCCAGCAGGACGTGGACGGGACCTACTTCCTGTACAGCAAGT TCTCGGTGGACA AGCCCAGCTGGGAGGGTGGAGGCATATTCCAGTGTGCGGTGATCGAC GAGGGTCTGCAC AACCACTACACCCAGAAGTCTATCTCCAAGACTCCGGGTAAATGA (SEQ ID NO: 99) IgG2 GCCCCCAAGACCGCCCCATTGCTCTACCCTCTCCCCCGCTGCCCCAG APKTAPLVYPLAPCGRDTSGPNVALG U03780 GGAACGTCTG CLASSYFPEPVTVTWNSGALTSGVHT GCCCTAACGTGGCCTTGGGCTGCCTGGCCTCAAGCTACTTCCCCGAG FPSVLQPSGLYSLSSMVTVPASSLSS CCAGTGACCGTG KSYTCNVNHPATTTKVDKRVGTKTKP ACCTGGAACTCGGGCGCCCTGACCACTGGGGTCCATACCTTCCCATC PCPICPACESPGPSVFIFPPKPKDTLMI CGTCCTCCACCG SRTPQVTCVVVDVSQENPEVQFSWYV GTCAGGGCTCTACTCCCTCAGCAGCATGGTGACCGTGCCGGCCAGCA DGVEVHTAQTRPKEEQFNSTYRVVSV GCCTCTCCACCA LIIQHQDWLNGKEFKCKVNNKDLPAPI AGAGCTACACCTGCAATGTCAACCACCCGGCCAGCACCACCAAGGTG TRISKAKGQTREPQVYTLPPHEELS GACAAGCCTCTT RSKVSITCLVIGFYPPDIDVEWQRNGQ GGAACAAAGACCAAAGCACCATGTCCCATATGCCCAGCCTGTCAATCG PEPEGNYRTTPPQQDVDGTYFLYSKF CCAGGGCCCTC SVDKASWQGGGIFQCAVMHEALHNHY GGTCTTCATCTTCCCTCCAAAACCCAACGACACCCTCATGATCTCCC TQKSISTPGK* CCACACCCCAGG (SEQ ID NO: 100) TTCAGGTGCGTGGTAGTTGATGTGAGCCAGGAGAAGGCGGAGGTCCA GTTCTCCTGGTAC GTGGACGGCCTAGAGGTGCACACCGCCCAGACGAGGCCAAAGGAGGA GCAGTTCAACAC CAGCTACCGCCTGGTCAGCGTCCTGCCCATCCAGCACGAGGACTGGCT GAACGGGAAGG AGTTCAAGTGCAAGGTCAACAACAAAGACCTCCCAGCCCCCATCACAA GGATCATCTCC AAGGCCAAAGGGCAGACCCGGGAGCCGCACGTGTACACCCTGCCCCCA CACCCCGAGG AGCTCTCCAGGACCAAAGTCACCATAACCTGCCTGCTCATTGCCTTCT ACCCACCTGAC ATCGATGTCGAGTGGCAAAGAAACGGACAGCCGGAGCCAGAGGGCAAT TACCGCACCAC CCCCCCCCAGCAGCACCTGGACGTGACCTACAACCTGTACAGCAAGT TCTCGGCGACA TAGGCCACCTGGCACGGTGGACGCATATTGGAGTGTTCCGGTGATGC ACGAGGCTCTGCAC AACCACTACACCCAGAAGTCTATCTCCAAGACTCCGGGTAAATGA (SEQ ID NO: 101) IgG3 GCCTACAACACACCTCCATCCGTCTACCCTCTGGCCCCCTGTGGCAGC AYNTAPSVYPLAPCGRDVSDHNVALG EU372858 CGGCGTCTCTCA CLVSSYFPEPVTVTWNSGALSRVVHT TCATAACGTGGCCTTGGGCTGCCTTGTCTCAAGCTACTTCCCCGAGC FPSVLQPSGLYSLSSMVIVAASSLSTL CAGTGACCGTGA SYTCNVYHPATNTKVDKRVDIEPPTPI CCTGGAACTCGGGTGCCCTGTCCAGAGTCGTGCATACCTTCCCATCC CPEICSCPAAEVLGAPSVFLFPPKPKD GTCCTGCAGCCG ILMISRTPKVTCVVVDVSQEEAEVQFS TCAGGCCTCTACTCCCTGAGCAGCATCGTGATCGTCCCGCCCACCAGC WYVDGVQLYTAQTRPMEEQFNSTYRV CTGTCCACCCT VSVLPIQHQDWLKGKEFKCKVNNKDLL GAGCTACACGTGCAACGTCTACCACCCGGCCACCAACACCAAGGTGGA SPITRTISKATGPSRVPQVYTLPPAWE CAAGCGTCTTG ELSKSKVSITCLVTGFYPPDIDVEQWS ACATCGAACCGCCCACACCCATCTGTCCCGAAATTTGCTCATGCCCAG NGQQEPEGNYRTTPPQQDVDGTYFLY CTGCAGAGGTC SKLAVDKVRWQRGDLFQCAVMHEALH CTCGGAGCACCGTCGGTCTTCCTCTTCCCTCCAAAACCCAAGGACATC NHYTQKSISKTQGK CTCATGATCTC (SEQ ID NO: 102) CCGGACACCCAAGGTCACGTGCGTCGTCGTGGACGTGAGCCAGGAGGA GGCTGAAGTCC AGTTCTCCTGGTACGTGGACGGCGTACAGTTGTACACGGCCCAGACG AGGCCAATGGAG GAGCAGTTGAACAGCACCTACCGCGTGGTCAGCGTCCTGCCCATCCAG CACCAGGACTG GCTGAAGGGGAACGAGTTCAAGTCCAAGGTCAACAACAAAGACCTCCT TTCCCCCATCA CGAGGACCATCTCCAAGGCTACAGGGCCGAGCCGGGTGCCGCAGGTGT ACACCCTGCC CCCAGCCTCCGAACACCTGTCCAACAGCAAAGTCACCATAACCTGCCT GGTCACTGGGT TCTACCCACCTGACATCGATGTCGAGTGGCAGAGCAACCGACAACAAG AGCCAGAGGGC AATTACCGCACCACCCCGCCCCAGCAGGAGGTGGATGGGACGTACTTC CTGTACAGCAA GCTCGCGGTGGACAAGGTCAGGTGGCAGCGFGGAGACCTATTCCAGTG TGCGGTGACGC ACGAGGCTCTGCACAACCACTACACCCAGAAGTCCATCTCCAAGACT CAGGGTAAATGA (SEQ ID NO: 103) IgG4 ACCTTCCCATCCGTCGTGCAGCCGTCAGGCCTCTACTCCCTCAGCAGC TFPSVLQPSGLYSLSSMVTVPASSLS U03782 ATGGTGACCGT SKSYTCNVNHPATTTKVDKRVGTKTK GCCGGCCAGCAGCCTGTCCAGCAAGAGCTACACCTGCAATGTCAACC PPCPICPACEGPGPSAFIFPPKPKDTL ACCCGGCCACCA MISTRPKVTCVVVDVSQENPEVQFSW CCACCAAGGTGGACAAGCGTGTTGGAACAAAGACCAAACCACCATGTC YVDGVEVHTAQTRPKEEQFNSTYRVV CCATATGCCCA SVLPIQHQDWLNGKEFKCKVNNKDLP GCCTGTGAAGGGCCCGGGCCCTCGGCCTTCATCTTCCCTCCAAAACCC APITRISKAKGQTREPQVYTLPPPTEE AAGGACACCCT LSRSKVTLTCLVTGFYPPDIDVEWQRN CATGATCTCCCGGACCCCCAAGGTCACGTGCGTGGTGGTAGATGTGAG GQPEPEGNYRTTPPQQDVDGTYFLYS CCAGGAGAACC KLAVDKASWQRGDTFQCAVMHEALH CGGAGGTCCAGTTCTCCTGGTACGTGGACGGCGTAGAGGTGCACACG NHYTQKSIFKTPGK* GCCCAGACGAGG (SEQ ID NO: 104) CCAAAGGACGAGCACTTCAACAGCACCTACCCCGTGGTCAGCGTCCTC CCCATCCACCA CCAGGACTGGCTGAACGGGAAGGAGTTCAAGTGCAAGGTCAACAACA AAGACCTCCCAG CCCCCATCACAAGGATCATCTCCAAGGCCAAAGGGCAGACCCGGGAGC CGCAGGTGTAC ACCCTGCCCCGACCCACCGAGGAGCTCTCCAGGTCCAAAGTCACGCTA ACCTGCCTGGT CACTGGCTTCTACCCACCTGACATCGATGTCGAGTGGCAAAGAAACGG ACAGCCGGAGC CAGAGGGCAATTACCGCACCACCCCGCCCCAGCAGGACGTGGACGGGA CCTACTTCCTG TACAGCAAGCTCGCCGTCCACAAGGCCACCTGCCAGCCTGGACACACA TTCCAGTGTCC GGTGATGCACGAGGCTCTGCACAACCACTACACCCAGAAGTCCATCTT CAAGACTCCGG GTAAATGA (SEQ ID NO: 105) IgG4 GCCCCCAAGACGGCCCCATCGGTCTACCCTCTGGCCCCCTGCGGCAGG APKTAPSVYPLAPCGRDVSGPNVALG EU372654 GACGTGTCTG CLASSYFPEPVTVTWNSGALTSGVHT GCCCTAACGTGGCCTTGGGGTGCCTGGCCTCAAGCTACTTCCCCGAGC FPSVLQPSGLYSLSSMVTVPASSLSS CAGTGACCGTG KSYTCNVNHPATTTKVDKRVGIHQPQ ACCTGGAACTCGGGCGCCCTGACCAGTGGCGTGCACACCTTCCCATCC TCPICPACEGPGPSAFIFPPKPKDTLMI GTCCTGCAGCC SRTPKVTCVVVDVSQENPEVQFSWYV GTCAGGGCTCTACTCCCTCAGCAGCATGGTGACCGTGCCGGCCAGCAG GHVEVHTAQTRPKEEQFNSTYRVVSV CCTGTCCAGCA LLIQHQDWLNGKEFKCKVNNKDLPAPI AGAGCTACACCTGCAATGTCTAACCAGCCGGCCACCACCACCAAGGTG TRISKAKGQTREPQVYTLPPPTEELSF GACAAGCGTGTT SKVTLTCLVTGFYPPDIDVEWQRNGQ GGAATACACCAGCCGCAAACATGTCCCATATGCCGAGCCTGTGAAGGG PEPEGNYRTTPPQQDVDGTYFLYSKL CCCCGGCCCTC AVDKASWQRGDTFQCAVMHEALHNH GGCCTTCATCTTCCCTCCAAAACCCAAGGACACCCTCATGATCTCCCG YT GACCCCCAAGG (SEQ ID NO: 106) TCACGTGCGTGGTGGTTGATGTGAGGCAGGAGAACCCGGAGGTCCAGT TGTCCTGGTAC GTGGACGGCGTAGAGGTGCACACGGCCCAGACGAGGCCAAAGGAGGAG CAGTTCAACAG CACCTACCGCGTGGTCAGCGTCCTGCTCATCCAGCACCAGGACTGGCT GAAGGGGAAGG AGTTCAAGTGCAAGCTCAAGAACAAAGACCTCCCAGCCCCCATCACAA GGATCATCTCC AAGGCCAAAGGGCAGACCCGGGAGCCGCAGGTGTACACCCTGCCCCCA CCCACCGAGG AGCTGTCCAGGAGCAAAGTCACGCTAACCTGCCTGGTCACTGGCTTCT ACCCACCTGAC ATCGATGTCGAGTGGCAAAGAAACGGACAGCCGGACCCAGAGGGCAAT TACCCCACCAC CCCGCCCCAGCAGGACGTGGACGGGACCTACTTCCTGTACAGCAAGCT CGCGGTGGACA AGGCCAGCTGGCAGCGTGGAGACACATTCCAGTGTGCGGTGATGCACG AGGCTCTGCAC AACCACTACACCC (SEQ ID NO: 107) IgG5 GCCCCCAAGACGGCCCCATCGGTCTACCCTCTGGCCCCCTGCAGCAG APKTAPSVYPLAPCSRDTSGPNVALG EU372657 GGACACGTCTG CLVSSYFPEPVTVTWNSGALTSGHVT GCCCTAACGTGGCCTCGGCTGCCTGGTCTCAAGCTACTTCCCCGAGC FPSVLQPSGLYSLSSMVTVPAHSLSS CAGTGACCGTG KRYTCNVNHPATKTKVDLCVGRPCPI ACCTGGAACTCGGGCGCCCTGACCAGTGGCGTGCACACCTTCCCATCC CPGCEVAGPSVFIFPPKPKDILMISRTP GTCCTGCAGCC EVTCVVVDVSKEHAEVQFSWYVDGEE CTCAGGGCTCTACTCCCTCAGCAGCATGGTGACCGGCCGGCCCACAGC VHTAETRPKEEQFNSTYRVVSVLPIQH TTGTCCAGCA EDWLKGKEFECKVNNEDLPGPITRTIS AGCGCTATACGTGCAATGTCAACCACCCAGCCACCAAAACCAAGCTGG KAKGVVRSPEVYTLPPPAEELSKSIVT ACCTGTGTGTT LTCLVKSIFPIFIHVEWEINGKPEPENA GGACGACCATGTCCCATATGCCCAGGCTGTGAAGTGCCCGGGCCCTCG YRTTPPQEDEDRTYFLYSKLAVDKAR CTCTTCATCTT WDHGETFECAVMHEALNHNYTQKSIS CCCTCCAAAACCCAAGGACATCCTCATGATCTCCCGGACCCCCGAGGT KTQGK* CACGTGCGTGG (SEQ ID NO: 108) TGGTGGACGTCAGCAAGGAGCACGCCGAGGTCCAGTTCTCCTGGTACG TGGACGGCGAA GAGCTGCACACCGCCGAGACCAGGCCAAAGGACGAGCAGTTCAACACC ACCTACCGCGT GGTCAGCGTCCTGCCCATCCAGCACGAGGACTGGCTGAAGGGGAAGG AGTTCGAGTGCA AGGTCAACAACGAAGACCTCCCAGGCCCCATCACGAGGACCATCTCCA AGCCCAAACCC GTGGTACGGAGCCGGGAGGTGTACACCCTGCCGCCACCCGCCGAGGA GCTGTCCAAGA GCATAGTCACGCTAACCTGCCTGGTCAAAAGCATCTTCCCGTCTTTCA TCCATGTTGAGT GGAAAATCAACGGAAAAGGAGAGCCAGAGAACGCATATCGCACCACC GCGGCTCAGGAC CACCAGGACAGGACCTACTCCCTGTACAGCAAGCTCGCCGTGCACAA CGCAAGATGGGA CCATGGAGAAACATTTGAGTGTGCGGTGATGCACGAGGCTCTGCACA ACCACTACACCC AGAAGTCCATCTCGAAGACTCAGGGTAAATGA (SEQ ID NO: 109) IgG5 GCCTACAACACAGCTCGATCGGTCTACCCTCTGGCCCCCTGTGGCAGG AYNTAPSVYPLAPCGRDVSDHNVALG EU372686 GACGTGTCTGA CLVSSYFPEPVTVTWNWGAQTSGVHT TCATAACGTGGCCTTGGGCTGCCTGGTCTCAAGCTACTTCCCCGAGCC FPSVLQPSGLYSLSSTVTVPAHSLSSK AGTGACCGTGA CFTCNVNHPATTTKVDLCVGKKTKPR CCTGGAACTGGGGCGCCCAGACCAGTGGCGTGCACACCTTCCCATCCG CPICPGCEVAGPSVFIFPPKPKDILMIS TCCTGCAGCCG RTPEVTCVVVDVSKEHAEVQFSWYVD TCAGGGCTCTACTGCCTCAGCAGCAGGGTGAGCGTGGCGGCCCACAG GDDVHTAETRPKEEQFNSTYRVVSVL CTTGTCCAGCAA PIQHEDWLKGKEFECKVNNEDLPGPIT CTGCTTCACGTGCAATGTCAACCACCCCGCCACCACCACCAAGGTCGA RTISKAKGVVRSPEVYTLPPPAEELSK CCTCTGTGTTG SIVTLTCLVKSFFPPFIHVEWKINGKPE GAAAAAAGACCAAGCCTCGATGTCCCATATGCCCAGGCTGTGAAGTG PENAYRTTPPQEDEDGTYFLYSKFSVE GCCGGGCCCTCG KFRWHSGGIHCAVMHEALHNHYT GTCTTCATCTTCCCTCCAAAACCCAAGGACATCCTCATGATCTCCCG (SEQ ID NO: 110) GACCCCCGAGGT CACGTGCGTGGTCGTGCACGTCAGCAAGGAGCACGCCGAGGTCCAGTT CTCCTGCTACC TGGACGGCGAAGAGGTGCACACGGCCGAGACGAGACCAAAGGAGGAG CAGTTCAACAGC ACTTACCGCGTGGTCAGCCTCCTGCCCATCCAGCACGAGGACTGGCTG AAGGGGAAGGA CTTCCAGTGCAACGTCAACAACGAACACGTCCCACGCCCCACGACGA GCACCATCTCCA AGCCCAAAGGGGTGGTACGGAGCCCGGAGGTGTACACCCTGCCCCCA CCCGCCGAGGA GCTGTCCAAGAGCATAGTCACGCTAACCTGCCTGGTCAAAAGCTTCTT CCCGCCTTTCAT CGATGTTGAGTGGAAAATCAACGCAAAACCAGAGGCAGAGAACCCATA CCCCACCACCC CGCCCCAGGAGGACGAGGACGGGACCTACTTCCTGTACAGCAAGTTCT CGGTGGAAAAG TTCAGGTGGCACAGTGGAGGCATCCACTGTGCGGTGATGCACGAGGCT CTGCACAACCA CTAGACCC (SEQ ID NO: 111) IgG6 CCCCCCAAGACGCCCCCATTAGTCTACCCTCTGGCCCCCTGCGGCACG APKTAPSVYPLAPCGRDTSGPNVALG EU372655 GACACGTCTG CLASSYFPEPVTLTWNSGALTSGVHT GCCCTAACGTGGCCTTGGGCTGCCTGGCCTCAAGCTACTTCCCCGAGC FPSVLQPSGLYSLSSMVTVPASSLSS CAGTGACCCTG KSYTCNVNHPATTTKVDLCVGRPCPI ACCTGGAACTCGGGCGCCCTGACCAGTGGCGTGCATACCTTCCCATCC CPACEGPGPSVFIFPPKPKDTLMISRT GTCCTGCAGCC PQVTCVVVDVSQENPEVQFSWYVDG GTCAGCCCTGTACTCCCTCAGCAGCATGGTGACCGGCCGGCCAGCAGC VEVHTAQTRPKEQFNSTYRVVSVLPI CTGTCCAGCA QHEDWLKGKEFECKVNNKDLPAPITRII AGAGCTACACCTGCAATGTCAACCACCCGGCCACCACCACCAAGGTGG SKSKGPSREPQVYTLSPSAEELSRSKV ACCTGTGTGTT SITCLVTGFYPPDIDVEWKSNGQPEPE GGACGACCATCTCCCATATGCCCAGCCTGTGAAGGGCCCGGGCCCTCG GNYRTTPPQQDVDGTYFLYSKLAVDK GTCTTCATCTT ASWQRGDPFQCAVMHEALHNHT CCCTCCAAAACCCAAGGACACCCTCATGATCTCCCGGACACGCCAGGT (SEQ ID NO: 112) CACGTGCGTGG TGGTAGATGTGAGCCAGGAAAACCCGGAGGTCCAGTTCTCCTGGTAT GTGGACGGTGTA GAGGTGCACACGGCCCAGACGAGGCCAAAGGAGGCGCAGTTCAACAGC ACCTACCGTGT GGTCAGCGTCCTGCCCATCCAGCACGAGGACTGGCTGAAGGGGAAGGA GTTCGAGTGCA AGGTCAACAACAAAGACCTCCCAGCCCGCATGACAAGGATCATCTGC AAGACCAAAGGG CCGAGCCGGGAGCCACAGGTGTACACCCTGTCCGCATCCGCCGAGGAG CTGTCCAGGA GCAAAGTCAGCATAACCTGCCTGGTCACTGGCTTCTACCCACCTGACA TCGATGTCGAG TGGAACAGCAACGCACAGCCGGAGCCAGAGGGCAATTACCCCACCACC CCGCCCCAGC AGGACGTGGACGGGACCTACTTCCTGTACAGCAAGCTCGCGGTGGAC AAGGCCAGCTGG CAGCGTGTGAGACCCATTCCAGTGTGCGGTGATGCACGAGGCTCTGCA CAACCACTACACCC (SEQ ID NO: 113) IgG6 CCCCCCAAGACGCCCCCATCGGTCTACCCTCTCGCCCCCTGCCCCAC APKTAPSVYPLAPCGRDTSGPNVALG EU372653 CCACACCTCTC CLASSYFPEPVTVTWNSGALTSGVHT GCCCTAACGTGCCCTTGGGCTGCCTGGCCTCAAGCTACTTCCCCGAGC FPSVLQPSGLYSLSSTVTVPARSSSRK CAGTGACCGTG CFTCNVNHPATTTKVDLCVGRPCPIC ACCTGGAACTCGGGCGCCCTGACCAGTGGCGTGCACACCTTCCCATGC PACEGNGPSVFIFPPKPKTLMISRTP GTCGTGCAGCC EVTCVVVDVSQENEPEVQFSWYVDGEE GTCAGCCCTCTACTCCCTCAGCAGCACGGTGACCGTGCCGCCCAGGAG VHTAETRPKEEQFNSTRYVVSVLPIQH CTCGTCCAGAA QDWLKGKEFECKVNNDLPAPITRIISK ACTGCTTCACGTGCAATGTCAACCACCCGGCCACCACCACCAAGGTGG AKGPSREPQVYTLSPSAEELSRSKVSI ACCTGTGTGTT TLLVTGFYPPDIDVEWKSNGQPEPEG GGACGACCATGTCCCATATGCCCAGCCTGTGAAGGGAACGGGCCCTCG NYRSTPPQEDEDGTYFLYSKLAVDKA GTCTTCATGTT RLQSGGIHCAVMHEALHNHYTQKSISK CCCTCGAAAACCCAAGGACACCCTCATCATCTCCCGGACCCCCGAGCT T CACGTGGCTCC (SEQ ID NO: 112) TGGTAGATGTGAGCCAGGAAAACCCGGAGGTCCAGTTCTCCTGGTACG TGGACGGCGAA GAGGTGCACACGGCCGAGACGAGGCCAAAGGAGGAGCAGTTCAACAG CACCTACCGTGT GGTCAGCCTCCTGCCCATCCAGCACCAGGACTGGCTGAAGGGAAAGG AGTTCGAGTGCA AGCTCAACAACAAAGACCTCCCAGCCCCCATCACAAGGATCATCTCC AAGGCCAAAGGG CCGAGGCGGGAGCCGCAGGTGTACACCCTGTCCCCATCCGCCGAGGAG CTGTCCAGGA GCAAAGTCAGCATAACCTGCCTGGTCACTGGCTTCTACCCACCTGAC ATCGATGTCGAG TGCAAGAGCAACGGAGACCCGGAGCCAGAGGGCAATTACCGCTCCAC CCCGCCCCAGGA GGACGAGGACGGGACCTACTTCCTGTACAGCAAACTCGCGGTGGACA AGGCGAGGTTGC AGAGTGCAGGCATCCACTGTGCGGTGATGCACGAGGCTCTGCACAACC ACTACACCCAG AAGTCCATCCCAAGACT (SEQ ID NO: 115) Por- Ig FP312892 http://www. Schwartz cine kappa imgt.org/ J.C. Ig (CK) IMGT et al light variant reportoire/ Immuno- chain 1 index. genetics con- Ig CU604948 ghp?section 84, 303- stant kappa =locus 313 (2012) region (CK) Genes& PMID: variant reportoire= 22108543 2 genetable& species= Pig &group=IGLC Ig CU407669 http://www. lambda imgt.org/ (CL) IMGT variant reportoire/ 1 index. Ig CU467599 ghp?section lambda =locus (CL) Genes& variant reportoire= 2 genetable& species= Pig &group=IGKC Water Water IgG1? GAGCGGCGTGCACACCTTCCCGGCCGTCCTTCAGTCC SGVHTFPAVLQSSGLYSLSSTVTAPAS NW_ Not None buffalo bu- TCCGGGCTCTACTCTCTGAGCAGCACGGTGACCGCGC ATKSQTFTCNVAHPASSTKVDKAVVP 005690903 registered (Scientific ffalo CCCCCAGCGCCACAAAAAGCCAGACCTTCACCTGCAA PCRPKPCDCCPPPELPGGPSVFIFPPK Name: Ig CGTAGCCCACCCGGCCAGCAGCACCAAGGTGGACAAG PKDTLTISGTPEVTCVVVDVGHDPEV Bubalus havy GCTGTTGTTGCCCCATGCAGACCGAAACCCTGTGATTG KFSWFVDDVEVNTARTKPREEQFNSTY bubalis) chain CTGCCCACCCCCTGAGCTCCCCGGAGGACCCTCTGTC RVVSALPIQHNDWTGGKEFKCKVYNEG con- TTCATCTTCCCACCAAAACCCAAGGACACCCTCACAAT LPAPIVRTISRTKGQAREPQVYVLAPP stant CTCTGGAACTCCTGAGGTCACGTGTGTGGTGGTGGAC QDELSKSTVSITCMVTGFYPDYIAVEW (CJ1 GTGGGCCAGGATGACCCCGAGGTGAAGTTCTCCTGGT QKDGQPESEDKYGTTPPQLDSDGSYF CH3) TCGTCGACGATGTGGAGGTAAACACAGCCAGGACGAA LYSRLRVNKNSWQEGAYICVVMHE GCCAAGAGAGGAGCAGTTCAACAGCACCTACTCGCGTG (SEQ ID NO: 118) GTCAGCGCCCTGCCCATCCAGCACAACGACTGGACTG GAGGAAAGGAGTTCAAGTGCAAGGTCTACAATGAAGGC CTCCCAGCCCCCATCGTGAGGACCATCTCCAGGACCA AAGGGCAGGCCCGGGAGCCGCAGGTGTACGTCCTGGC CCGACCCCAGGACGAGCTCACCAAAAGCACGGTCAGC ATCACTTGCATGGTCACTGGCTTCTACCCAGACTACAT CGCCGTAGAGTGGCAGAAAGATGGGCAGCCTGAGTCA GAGGACAAATATGGCACGACCCCGCCCCAGCTGGACA GCGATGGCTCCTACTTCCTGTACAGCAGGCTCAGGGT GAACAAGAACAGCTGGCAAGAAGGAGGCGCCTACACG TGTGTAGTGATGCATGAGGC (SEQ ID NO: 119) IgG2? GCCTCCATCACAGCCCCGAAAGTCTACCCTCTGACTTC ASITAPKVYPLTSCRGETSSSTVTLGC NW_ TTGCCGCGCGGAAACGTCCAGCTCCACCGTGACCCTG LVSSYMPEPVTVTWNSGALKSGVHTF 005766143 GGCTGCCTGGTGTCCAGCTACATGCCCGAGCCGGTGA PAVLQSSGLYSLSSTVTAPASATKSQT CGGTGACCTGGAACTCGGGTGCCGTGAAGAGCGGCGT FTCNVAHPASSTKVDTAVGFSSDCCK GCACACCTTCCCGGCCGTCCTTCAGTCCTCTGGGCTC FPKPCVRPGSVFIFPPKPKDTLMITGNP TACTCTCTCAGCAGCACGGTGACCGCGCCCGCCAGCG EVTCVVDVGRDNPEVQFSWFVGDVE CCACAAAAAGCCAGACCTTCACCTGCAACGTAGCCCAC VHTGRSKPREEQFNSTRYVVSTLPIQH CCGGCCAGCAGCACCAAGGTGGACACGGCTGTTGGGT NDWTGGKEFKCKVNKGLPAPIVRTIS TCTCGAGTGACTGCTGCAAGTTTCCTAAGCCTTGTGTG RIIKGQAREPQVYLAPPQEELLSKSTVS AGGGGACCATCTGTCTTCATCTTCCCGCCGAAACCCAA VTCMVTGFYPDYIAVEWHRDRQAESED AGACACCCTGATGATCACAGGAAATCCCGAGGTCACAT KYRTTPPQLDSDGSYFLYSRLKVNKNS GTGTGGTGGTGGACGTGGGCCGGGATAACCCCGAGGT WQEGGAYTCVYMHE GCAGTTCTCCTGGTTCGTGGGTGATGTGGAGGTGCAC (SEQ ID NO: 120) ACGGGCAGGTCGAAGCCGAGAGAGGAGCAGTTCAACA GCACCTAGCGCGTGGTCAGCACCCTGCCCATCCAGCA CAATGACTGGACTGGAGGAAAGGAGTTCAAGTGCAAG GTCAACAACAAAGGCCTCCCAGCCCCCATCGTGAGGA CCATCTCCAGGACCAAAGGGCAGGCCCGGGAGCCGCA GGTGTACGTCCTGGCCCCACCCCAGGAAGAGCTCAGC AAAAGCACGGTCAGCGTCACTTCCATGGTCACTGGCTT CTACCCAGACTACATCGCCGTAGAGTCGCATAGAGACC GGCAGGCTGAGTCGGAGGACAAGTACCGCACGACCCC GCCCCAGCTGGACAGCGATGGCTCCTACTTCCTGTAC AGCAGGCTCAAGGTGAACAAGAACAGCTGGCAAGAAG GAGGCGCCTACACGTGTGTAGTGATGCATGAGGC (SEQ ID NO: 121) IgG3? GCCTCCACCACAGCCCCGAAAGTCTACCCTCTGGCAT ASTTAPKVYPLASSCGDTSSSTVTLGC NW_ CCAGCTGCGGGGACACGTCCAGGTCCACCGTGACCCT LVSSYMPEPVTVTWNSGALKNGVHTF 005784206 GGGCTGCCTGGTCTCCAGCTACATGCCCGAGCCGGTG PAVRQSSGLYSLSSMVTMPTSTAGTQ ACCGTGACCTGGAACTCGGGTGCCCTCAAGAACGGCG TFTCNVAHPASSTKVDTAVTARHPVP TGCACACCTTCCCGGCCGTCGGGCAGTCCTCCGGGCT KTPETPIHPVKPPTQEPRDEKTPCQCP CTACTCTCTCAGCAGCATGGTGACCATGCCCACCAGCA KCPEPLGGLSVFIFPPKPKDTLTISGTP CCGCAGGAACCCAGACCTTCACCTGCAACGTAGCCCA EVTCVVVDVGQDDPEVQFSWFVDDVE CCCGGCCAGCAGCACCAAGGTGGACACGGCTGTCACT VHTARMKPREEQFNSTYRVVSALPIQH GCAAGGCATCCGGTCCCGAAGACACCAGAGACACCTA QDWLREKEFKCKVNNKGLPAPIVRTISR TCCATCCTGTAAAACCCCCAACCCAGGAGCCCAGAGAT TKGQAREPQVYVLAPPREELSKSTLSL GAAAAGACACCCTGCCAGTGTCCCAAATGCCCAGAACC TCLITGFYPEEVDVEWQRNGQPESEDK TCTGGGAGGACTGTCTCTCTTCATCTTCCCACCGAAAC YHTTPPQLDADGSYFLYSRLRVKRSSW CCAAGGACACCCTCACAATCTCTGGAACGCCCGAGGT QEGDHYTCAVMHEALRNHYKEKPISRS CACGTGTGTGGTGGTGGACGTGGGCCAGGATGACCCC PGK* GAAGTGCAGTTCTCCTGGTTCGTGGATGACGTGGAGG (SEQ ID NO: 122) TGCACACAGCCAGGATGAAGCCAAGAGAGGAGCAGTT CAACAGCACCTACCGCGTGGTCAGCGCCCTGCCCATC CAGCACCAGGACTGGCTGCGGGAAAAGGAGTTCAAGT GCAAGGTCAACAACAAAGGCCTCCCGGCCCCCATCGT GAGGACCATCTCCAGGACCAAAGGGCAGGCCCGGGAG CCACAGGTGTATGTCCTGGCCCCACCCCGGGAAGAGC TCAGCAAAAGCACGCTCAGCCTCACCTGCCTAATCACC GGCTTCTACCCAGAAGAGGTAGACGTGGAGTGGCAGA GAAATGGGCAGCCTGAGTCAGAGGACAAGTACCACAC GACCCCACCCCAGCTGGACGCTGACGGCTCCTACTTC CTGTACAGCAGGCTCAGGGTGAACAGGAGCAGCTGGC AGCAAGGAGACCACTACACGTGTGCAGTGATGCATGAA GCTTTACGGAATCACTACAAAGAGAAGCCCATCTCGAG GTCTCCGGCTAAATCA (SEQ ID NO: 123) Water Ig CAGCCCAAGTCCGCACCCTCAGTCACCCTGTTCCCAC QPKSAPSVTLFPPSTEELSANKATLVG NW_ Not None bu- lambda? CCTCCACGGAGGAGCTCAGCGCCAACAAGGCCACCCT LISDFYPGSMTVARKADGSTITRNVETT 005690786 registered ffalo GGTGTGTCTCATCAGCGACTTCTACCCGGGTAGCATGA RASKQSNSKYAASSYLSLTGSEWKSKG Ig CCGTGGCCAGGAAGGCAGACGGCAGCACCATCACCCG SYSCEVTHEGSTVTKTVKPSECS* light GAACGTGGAGACCACCCGGGCCTCCAAACAGAGCAAC (SEQ ID NO: 116) chain AGCAAGTACGCGGCCACCAGCTACCTCAGCCTGACGG con- GCAGCGAGTGGAAATCGAAAGGCAGTTACAGCTGCGA stant GGTCACGCACGAGGGGAGCACCGTGACAAAGACAGTG region AAGCGCTCAGAGTGTTCTTAG (CL) (SEQ ID NO: 117)

The amino acid sequences as shown in SEQ ID NOS: 8 to 13, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120 and 122 may have deletion(s), substitution(s) or addition(s) of one or several (e.g., up to five, about 10 at the 3 most) amino acids. Even when such mutations have been introduced, the resulting amino acid sequences are capable of having the function as the constant region of Ig heavy chain or light chain.

The anti-PD-L1 antibody of the present invention may have a four-chain structure comprising two light chains and two heavy chains.

The anti-PD-L1 antibody of the present invention may be prepared as described below. Briefly, an artificial gene is synthesized which comprises the light chain sequence (variable region sequence and constant region sequence) and the heavy chain sequence (variable region sequence and constant region sequence) of the anti-PD-L1 antibody of the present invention. The resultant gene is inserted into a vector (e.g., plasmid), which is then introduced into a host cell (e.g., mammal cell such as CHO cell). The host cell is cultured, and the antibody of interest is collected from the resultant culture. In the synthesis of the artificial gene, codons of the nucleotide sequence may be optimized.

The present invention provides a DNA encoding an anti-PD-L1 antibody comprising (a) a light chain comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3). CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5). The present invention also provides a DNA encoding a light chain of an anti-PD-L1 antibody comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) (the DNA of (a′)). Further, the present invention provides a DNA encoding a heavy chain of an anti-PD-L1 antibody comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5) (the DNA of (b′)).

For (a) a light chain comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5), reference should be had to the foregoing description. A DNA comprising the DNA of (a′) and the DNA of (‘b) may be synthesized on commercial synthesizer. Restriction enzyme recognition sites, KOZAK sequences, poly-A addition signal sequences, promoter sequences, intron sequences or the like may be added to this DNA.

The present invention also provides a vector comprising the above-mentioned DNA encoding an anti-PD-L1 antibody.

As the vector, Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12 or pUC13); Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5 or pC194), yeast-derived plasmids (e.g., pSH19 or pSH15); bacteriophages such as λ phage; animal viruses such as retrovirus or vaccinia virus; or insect pathogen viruses such as baculovirus may be used. In the Examples described later, pDC6 (Japanese Patent No. 5704753, U.S. Pat. No. 9,096,878, EU Patent 2385115, Hong Kong (China) patent HK1163739 and Australia Patent 2009331326) is used.

The vector may also comprise promoters, enhancers, splicing signals, poly-A addition signals, intron sequences, selection markers, SV40 replication origins, and so forth.

The present invention also provides a host cell transformed by the above vector. It is possible to prepare the anti-PD-L1 antibody of the invention by culturing the host cell and collecting the antibody of interest from the resultant culture. Therefore, the present invention also provides a method of preparing an antibody, comprising culturing the above-described host cell and collecting the anti-PD-L1 antibody of the invention from the culture. In the method of the present invention for preparing an antibody, a vector incorporating a DNA comprising a DNA encoding the light chain and a DNA encoding the heavy chain may be transfected into a host cell. Alternatively, a vector incorporating a DNA encoding the light chain and a vector incorporating a DNA encoding the heavy chain may be co-transfected into a host cell.

Examples of the host cell include, but are not limited to, bacterial cells (such as Escherichia bacteria, Bacillus bacteria or Bacillus subtilis), fungal cells (such as yeast or Aspergillus), insect cells (such as S2 cells or Sf cells), animal cells (such as CHO cells, COS cells. HeLa cells. C127 cells, 3T3 cells. BHK cells or HEK 293 cells) and plant cells. Among these, CHO-DG44 cell (CHO-DG44(dfhr^(−/−))) which is a dihydrofolate reductase deficient cell is preferable.

Introduction of a recombinant vector into a host cell may be performed by the methods disclosed in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (e.g., the calcium phosphate method, the DEAE-dextran method, transfection, microinjection, lipofection, electroporation, transduction, scrape loading, the shotgun method, etc.) or by infection.

The resultant transformant may be cultured in a medium, followed by collection of the anti-PD-L1 antibody of the present invention from the culture. When the antibody is secreted into the medium, the medium may be recovered, followed by isolation and purification of the antibody from the medium. When the antibody is produced within the transformed cells, the cells may be lysed, followed by isolation and purification of the antibody from the cell lysate.

Examples of the medium include, but are not limited to, OptiCHO medium, Dynamis medium, CD CHO medium, ActiCHO medium. FortiCHO medium, Ex-Cell CD CHO medium. BalanCD CHO medium, ProCHO 5 medium and Cellvento CHO-100 medium.

The pH of the medium varies depending on the cell to be cultured. Generally, a pH range from 6.8 to 7.6 is used; mostly, a pH range from 7.0 to 7.4 is appropriate.

When the cell to be cultured is CHO cells, culture may be performed by methods known to those skilled in the art. For example, it is usually possible to perform culturing in a gas-phase atmosphere having a CO₂ concentration of 0-40%, preferably 2-10%, at 30-39° C., preferably around 37° C.

The appropriate period of culture is usually from one day to three months, preferably from one day to three weeks.

Isolation and purification of the antibody may be performed by known methods. Known isolation/purification methods which may be used in the present invention include, but are not limited to, methods using difference in solubility (such as salting-out or solvent precipitation); methods using difference in molecular weight (such as dialysis, ultrafiltration, gel filtration or SDS-polyacrylamide gel electrophoresis); methods using difference in electric charge (such as ion exchange chromatography); methods using specific affinity (such as affinity chromatography); methods using difference in hydrophobicity (such as reversed phase high performance liquid chromatography): and methods using difference in isoelectric point (such as isoelectnc focusing).

It is also possible to prepare the anti-PD-L1 antibody of the present invention by culturing a hybridoma which may be prepared by the method disclosed in the literature (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y. Murata S, Ohashi K, Immunology. 2014 August; 142(4):551-61). A hybridoma producing anti-PD-L1 antibody 6C11-3A11 is stored at the laboratory of the present inventors (Laboratory of Infectious Diseases, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University).

The PD-L1 antibody of the present invention may be used for detecting PD-L. Therefore, the present invention provides a composition for detecting PD-L1, comprising the PD-L1 antibody as an active ingredient.

Detection of PD-L1 may be performed by such methods including, but are not limited to, immunohistochemical staining, immunocytochemical staining, flow cytometry, enzyme linked immunosorbent assay (ELISA) and Western blotting.

Analytes for detection may be exemplified by samples such as tissues or body fluids taken from organisms (e.g., blood (whole blood, plasma, serum, or specific cell such as erythrocyte, leukocyte or lymphocyte), urine, saliva, etc.); cell culture; and cultured cells (established cell lines, primary cultured cells, subcultured cells, etc.). The source of such analytes is not particularly limited. Examples include rat, canine, ovine, goat, porcine, feline, human, equine, bovine, water buffalo, yak, rabbit, mouse, hamster, and guinea pig.

The PD-L1 antibody of the present invention may be labeled with radioisotopes, enzymes, luminescent substances, fluorescent substances, biotin, or the like. If reaction with a primary antibody (the anti-PD-L1 antibody of the present invention) which specifically binds to a target molecule (PD-L1) is followed by reaction with a secondary antibody which binds to the primary antibody so as to detect the target molecule, it is suitable to label the secondary antibody.

Since PD-L1 is strongly expressed in cancer cells and virus-infected cells, the composition of the present invention may be used for diagnosis of cancers and/or infections. Usually, the amount (concentration) of PD-L in an analyte is determined based on the amount (concentration) of the complex of PD-L1 and anti-PD-L1 antibody. When the amount (concentration) of PD-L1 in the analyte is high compared to negative control (e.g., healthy surrounding tissue (connecting tissue, blood vessels, etc.)), the analyte may be diagnosed as suffering cancer and/or infection. Alternatively, if PD-L1 is detected in the analyte, the analyte may be diagnosed as suffering cancer and/or infection.

Examples of cancers and/or infections include, but are not limited to, neoplastic diseases (e.g., malignant melanoma, lung cancer, gastric cancer, renal cancer, breast cancer, bladder cancer, esophageal cancer, ovarian cancer and the like), leukemia, Johne's disease, anaplasmosis, bacterial mastitis, mycotic mastitis, mycoplasma infections (such as inycoplasma mastitis, mycoplasma pneumonia or the like), tuberculosis, Theileria orientalis infection, cryptosporidiosis, coccidiosis, trypanosomiasis and leishmaniasis.

The composition of the present invention can be used to select subject animals suitable for therapy using an anti-PD-L1 antibody. For example, animals satisfying the following two points may be considered as candidate animals.

1. A case diagnosed as suffering cancer (such as melanoma) or infection in pathological examination

2. A case found positive for anti-PD-L1 antibody

Negative control may be healthy surrounding tissue (connecting tissue, blood vessels, etc.), and positive control may be a case of cancer (such as melanoma) or infection. Basically, animals with a tumor which is positive in immunohistochemical staining of almost all regions may be subjected to clinical trial.

Subject animals are not particularly limited and may include rat, canine, ovine, goat, porcine, feline, human, equine, bovine, water buffalo, yak, rabbit, mouse, hamster, and guinea pig.

The composition of the present invention may further comprise reagents for detecting labels, diluents, lavage fluids, written instructions describing criteria for diagnosis/selection, and so on.

EXAMPLES

Hereinbelow, the present invention will be described in more detail with reference to the following Examples. However, the present invention is not limited to these Examples.

Example 1

1. Introduction

Programmed cell death 1 (PD-1), an immunoinhibitory receptor, and its ligand programmed cell death ligand 1 (PD-L1) are molecules identified by Prof. Tasuku Honjo et al., Kyoto University, as factors which inhibit excessive immune response and are deeply involved in immunotolerance. Recently, it has been elucidated that these molecules are also involved in immunosuppression in infections and tumors in various animals. In the subject Example, an anti-bovine PD-L1 monoclonal antibody was prepared by immunizing rats, and then a clone (6C11-3A11) capable of detecting canine PD-L1 was selected. Further, the present inventors performed immunohistochemical staining to examine whether or not this anti-bovine PD-L1 antibody 6C11-3A11 would be useful for detecting PD-L1 in canine malignant tumors (such as melanoma) and porcine/ovine infections.

2. Materials and Methods

2.1 Rat Anti-Bovine PD-L1 Monoclonal Antibody Producing Cells

The nucleotide sequence of bovine PD-L1 was identified (Ikebuchi R, Konnai S, Shirai T. Sunden Y, Murata S. Onuma M, Ohashi K. Vet Res. 2011 Sep. 26:42:103). Based on the sequence information, a recombinant bovine PD-L1 was prepared. Rat was immunized in the footpad with this recombinant protein, and hybridomas were established by the iliac lymph node method. As a result, a plurality of hybridomas producing rat anti-bovine PD-L1 monoclonal antibodies were obtained (Ikebuchi R, Konnai S, Okagawa T, Yokoyama K, Nakajima C, Suzuki Y, Murata S, Ohashi K. Immunology 2014 August; 142(4):551-561). Rat anti-bovine PD-L1 antibody 6C11-3A11 is one of the monoclonal antibodies established from the above-described immunized rat.

2.2 Identification of Full-Length Canine PD-L1 Gene

To determine the full length of canine PD-L1 cDNA, PCR primers were first designed based on the putative nucleotide sequence of canine PD-L1 already registered at The National Center for Biotechnology Information (NCBI) (GenBank accession number; XM_541302). Briefly, primers to amplify the inner sequence of the open reading frame (ORF) of this gene were designed (cPD-1 inner F and R), and PCR was performed. For the amplified products, nucleotide sequences were determined with a capillary sequencer according to conventional methods. Further, to determine the nucleotide sequence of full-length PD-L1 cDNA, primers (cPD-L1 5′ GSP and 3′GSP) were designed based on the canine PD-L1 cDNA sequences determined above. 5′-RACE and 3-RACE were then performed using, respectively, the 5′-RACE system for rapid amplification of cDNA ends and 3′-RACE system for rapid amplification of cDNA ends (Invitrogen). The resultant gene fragments of interest were sequenced as described above (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y. Nakajima C, Suzuki Y. Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415).

Primer (cPD-L1 inner F):  (SEQ ID NO: 22) ATGAGAATGTTTAGTGTCTT Primer (cPD-L1 inner R):  (SEQ ID NO: 23) TTATGTCTCTTCAAATTGTATATC Primer (cPD-L1 5′GSP): (SEQ ID NO: 24) TTTTAGACAGAAAGTGA Primer (cPD-L1 3′GSP): (SEQ ID NO: 25) GACCAGCTCTTCTTGGGGAA 2.3 Preparation of Canine PD-L1 Expressing COS-7 Cells

For preparing a canine PD-L1-EGFP expression plasmid, PCR was performed using a synthesized beagle PBMC-derived cDNA as a template and primers designed by adding BglII and EcoRI recognition sites on the 5′ side (cPD-L1-EGFP F and R). The resultant PCR products were digested with BglII (New England Biolabs) and EcoRI (Takara), and then purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics), followed by cloning into pEGFP-N2 vector (Clontech) similarly treated with restriction enzymes. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pEGFP-N2-cPD-L1.

Primer (cPD-L1-EGFP F): (SEQ ID NO: 26) GAAGATCTATGAGAATGTTTAGTGTC Primer (cPD-L1-EGFP R): (SEQ ID NO: 27) GGAATTCTGTCTCTTCAAATTGTATATC

COS-7 cells were subcultured at a density of 5×10⁴ cells/cm in 6-well plates, and then cultured overnight in RPMI 1640 medium containing 10% inactivated fetal bovine serum and 0.01% L-glutamine at 37° C. in the presence of 5% CO₂. The pEGFP-N2-cPD-L1 or pEGFP-N2 (negative control) was introduced into COS-7 cells at 0.4 μg/cm² using Lipofectamine 2000 (Invitrogen). The cells were cultured for 48 hours (canine cPD-L-EGFP expressing cell and EGFP expressing cell). In order to confirm the expression of PD-L1 in the thus prepared expressing cells, intracellular localization of enhanced green fluorescent protein (EGFP) was visualized with an inverted confocal laser microscope LSM700 (ZEISS) (Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M. Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K. PLoS One. 2014 Jun. 10; 9(6):e98415).

2.4 Cross-Reactivity of Rat Anti-Bovine PD-L1 Antibody 6C11-3A11 with Canine PD-L1

In order to confirm that rat anti-bovine PD-L1 antibody 6C1-3A11 specifically binds to canine PD-L1, flow cytometry was performed using the canine cPD-L1-EGFP expressing cell or the EGFP expressing cell prepared in 2.3 above. To 2×10⁵-1×10⁶ cells, 10 μg/ml of anti-bovine PD-L1 antibody 6C11-3A11 was added and reacted for 30 min at room temperature. After washing, the antibody binding to cell surfaces was detected with Allophycocyanine-labeled goat anti-rat Ig antibody (Beckman Coulter). For the analysis, FACS Verse (Becton. Dickinson and Company) was used. As a negative control antibody, rat IgG2a (κ) isotype control (BD Bioscience) was used. For every washing operation and dilution of antibodies, 10% inactivated goat serum-supplemented PBS was used. The results are shown in FIG. 1.

2.5 CDR Analysis of Rat Anti-Bovine PD-L1 Antibody 6C1-3A11

The heavy chain and the light chain genes of rat anti-bovine PD-L1 antibody 6C11-3A11 were identified from a hybridoma producing the antibody by RACE method. The complementarity-determining regions (CDRs) of rat anti-bovine PD-L1 antibody 6C11-3A11 were determined using NCBI IGBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The results are shown in FIG. 2.

2.6 Immunohistochemical Staining of Canine Tumor Tissues and Ovine/Porcine Infected Tissues

In order to confirm that rat anti-bovine PD-L1 antibody 6C11-3A1 is applicable to PD-L1 immunohistochemical staining of canine tumors, formalin-fixed and paraffin-embedded canine tumor samples were immunohistochemically stained. According to conventional methods, the resultant samples were deparaffinized and then subjected to microwave treatment (5 min, twice) in citrate buffer. Subsequently, the samples were reacted with PD-L1 antibody 6C11-3A11 (400-fold dilution) for 30 min and then with Simple Stain Mouse MAX-PO (Rat) (Nichirei Bioscience) for 30 mm. For coloring, diaminobenzidine (DAB) was reacted for 10 min.

The results are shown in FIGS. 3, 4, 5-1, 5-2, 6 and 7.

Anti-MelanA antibody, the only commercially available antibody specific to melanoma, stained tumor cells very weakly (FIG. 3, left). On the other hand, the PD-L1 antibody (6C11-3A11) established by the present inventors stained tumor cells very strongly (FIG. 3. Right). The PD-L1 antibody (6C11-3A11) was capable of staining almost all cases of melanoma.

In canine melanoma, tumor cells were found diffusely positive for the PD-L1 antibody (6C1-3A11). (Positive number/tested number=12/12: positive rate 100%)

In canine lymphoma, tumor cells were found diffusely positive for the PD-L1 antibody (6C11-3A11). In canine osteosarcoma, some tumor cells were stained intracellularly. In canine renal cell carcinoma, tumor cells were found diffusely positive in various tissue types.

In a case of ovine listeriosis, a PD-L1 staining image of a brain lesion of ovine listeriosis showing neurologic symptoms is shown in FIG. 6, left panel. In an enlarged photograph of this image, expression of PD-L1 was observed in macrophages infiltrating into brain lesions (FIG. 6, Right).

In a case of porcine circovirus type 2 infection, PD-L1 was stained with lymphoid follicles, and virus was stained in these cells (FIG. 7, Left).

In a case of porcine mycoplasma pneumonia, a great number of macrophages infiltrated pulmonary lesions, and PD-L1 was stained in these infiltrating cells (FIG. 7, Right).

As described so far, anti-bovine PD-L1 antibody 6C11-3A11 may be used for detecting PD-L1 in various canine tumors (such as malignant melanoma) and ovine/porcine infections by immunohistochemical staining. This suggests the possibility of using anti-bovine PD-L1 antibody 6C11-3A11 for diagnosis in a multiple-animal and a multiple-disease manner.

Example 2J

1. Introduction

Monoclonal antibodies may be produced by culturing hybridomas and purifying antibodies from the resultant culture supernatants. Alternatively, when the nucleotide sequence of an antibody of interest has been identified, a cell expressing the antibody may be prepared by transfecting cultured cells with a vector for expressing the nucleotide sequence; the thus prepared antibody expressing cell may be used as a substitute for hybridoma. In the subject Example, a method will be illustrated below in which an antibody is produced by a protein expression system using an expression vector and a mammalian cell.

2. Materials and Methods

2.1 Preparation of Rat Anti-Bovine PD-L1 Antibody 6C11-3A1 Expression Vector

Based on the nucleotide sequence of rat anti-bovine PD-L1 antibody 6C1-3A11 identified in 2.5 of Example 1 above, gene synthesis is performed so that NotI restriction enzyme recognition site, KOZAK sequence, antibody's light chain sequence, poly-A addition signal sequence (PABGH), promoter sequence (PCMV), SacI restriction enzyme recognition site, intron sequence (INRBG), KOZAK sequence, antibody's heavy chain sequence and XbaI restriction enzyme recognition site will be located in this order. In this case, codons of the antibody gene may have been optimized in advance depending on the type of the cell that is to express the gene. The synthesized gene strand is incorporated into an expression vector pDC6 (kindly provided by Prof. S. Suzuki, Research Center for Zoonosis Control, Hokkaido University) at the cloning site (NotI and XbaI restriction enzyme recognition sequences located downstream of PCMV and between INRBG and PABGH) using restriction enzyme recognition sequences so that the above-listed sequences will be located in the above-mentioned order to thereby construct a rat anti-bovine PD-L1 antibody expressing vector pDC6.

2.2 Expression of Rat Anti-Bovine PD-L1 Antibody 6C11-3A11

The rat anti-bovine PD-L1 antibody expressing vector pDC6 as prepared in 2.1 above is transfected into CHO-DG44 cells (CHO-DG44(dfhr^(−/−))) which are dihydrofolate reductase deficient cells, and high expression clones are selected by dot blotting. For increased expression, gene amplification treatment may be performed by adding load on cells in a medium containing 60 nM, 250 nM or 1000 nM methotrexate (Mtx). The thus prepared cells stably expressing rat anti-bovine PD-L1 antibody 6C11-3A11 are transferred to Mtx-free Opti-CHO medium. By culturing those cells under shaking for 14 days (125 rpm, 37° C., 5% CO₂), a culture supernatant containing the antibody of interest can be obtained. The antibody in the culture supernatant may be purified by known methods such as affinity chromatography or ion exchange chromatography for use in various experiments.

Example 3

1. Introduction

For the purpose of establishing a novel diagnosis method for tumor diseases, a rat-human chimeric anti-PD-L1 antibody is obtained in the subject Example by culturing Chinese hamster ovary cells (CHO cells) that will express a chimeric antibody gene in which the variable region gene of rat anti-bovine PD-L antibody 6C11-3A11 is combined with the constant region gene of human immunoglobulin (IgG4).

2. Materials and Methods

2.1 Preparation of Rat-Human Chimeric Anti-PD-L1 Expression Vector (FIG. 10)

Hereinbelow, a rat-human chimeric anti-PD-L1 antibody is established using rat anti-bovine PD-L1 monoclonal antibody 6C1-3A11 as its variable region.

Briefly, heavy chain and light chain variable region genes were identified from a hybridoma producing the rat anti-bovine PD-L1 antibody 6C11-3A11. Further, a nucleotide sequence was prepared by linking the heavy chain and light chain variable region genes of the above rat antibody to the constant region of heavy chain IgG4 and the constant region of light chain Kappa of a known human antibody, respectively. After codon optimization, gene synthesis is performed so that NotI restriction enzyme recognition site, KOZAK sequence, chimeric antibody's light chain sequence, poly-A addition signal sequence (PABGH), promoter sequence (PCMV), SacI restriction enzyme recognition site, intron sequence (INRBG), KOZAK sequence, chimeric antibody's heavy chain sequence and XbaI restriction enzyme recognition site will be located in this order. The synthesized gene strand is incorporated into the expression vector pDC6 (kindly provided by Prof. S. Suzuki, Research Center for Zoonosis Control, Hokkaido University) at the cloning site (NotI and XbaI restriction enzyme recognition sequences located downstream of PCMV and between INRBG and PABGH) using restriction enzyme recognition sequences so that the above-listed sequences will be located in the above-mentioned order (FIG. 10). Thus, a rat-human chimeric anti-PD-L1 antibody expressing vector is constructed. This expression vector is transfected into CHO-DG44 cells (CHO-DG44(dfhr^(−/−))) which are dihydrofolate reductase deficient cells, and high expression clones are selected by dot blotting. For increased expression, gene amplification treatment may be performed by adding load on cells in a medium containing 60 nM, 250 nM or 1000 nM methotrexate (Mtx). The thus prepared cells stably expressing rat-human chimeric anti-PD-L1 antibody 6C11-3A11 are transferred to Mtx-free Opti-CHO medium. By culturing those cells under shaking for 14 days (123 rpm, 37° C., 5% CO₂), a culture supernatant containing the antibody of interest can be obtained. The antibody in the culture supernatant may be purified by known methods such as affinity chromatography or ion exchange chromatography for use in various experiments.

Example 41

1. Introduction

With respect to PD-L1 in canine tumors, a detection method by immunohistochemical staining with rat anti-bovine PD-L1 antibody 6G7-E1 was previously established, and the expression profiles in various tumors have been reported (Maekawa N, Konnai S, Okagawa T, Ikebuchi R, Izumi Y, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Kato Y, Murata S. Ohashi K. PLoS One. 2016 Jun. 11(6): e0157176). In the subject Example, in order to examine whether rat anti-bovine PD-L1 antibody 6C11-3A11 is more useful than existing anti-PD-L1 antibody 6G7-E1 in expression analysis of PD-L1 in canine tumors, immunohistochemical staining of various canine tumors was performed to thereby directly compare the PD-L1 detection sensitivities of 6G7-E1 and 6C11-3A11.

2. Materials and Methods

2.1 Comparison by Flow Cytometry Using Canine PD-L-EGFP Stably Expressing CHO-DG44 Cells (FIG. 11)

First, in order to prepare canine PD-L1 membrane expressing cells, 2.5 μg of canine PD-L1-EGFP expression plasmid (pEGFP-N2-cPD-L1) prepared in 2.3 of Example 1 or pEGFP-N2 (negative control) was introduced into 4×10⁶ CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing G418 (Enzo Life Science) 800 μg/ml, GlutaMAX supplement (Life Technologies) 20 ml-L, and 10% Pluronic F-68 (Life Technologies) 18 ml/L, followed by selection of stably expressing cells and cloning by limiting dilution. The thus prepared canine PD-L1 membrane expressing cell or EGFP expressing cell was reacted with rat anti-bovine PD-L1 antibody 6C11-3A11 or 6G7-E1 at room temperature for 30 min. After washing, antibodies binding to cell surfaces were detected with Allophycocyanine-labeled goat anti-rat Ig antibody (Beckman Coulter). For analysis, FACS Verse (Becton. Dickinson and Company) was used. As a negative control, rat IgG2a (κ) or IgM (κ) isotype control (BD Bioscience) was used. For every washing operation and dilution of antibodies, 10% inactivated goat serum-supplemented PBS was used.

The results are shown in FIG. 11. Rat anti-bovine PD-L1 antibodies 6C11-3A11 and 6G7-E1 bound specifically to canine PD-L1 membrane expressing cells. The resultant fluorescence intensity was higher with 6C11-3A11 than with 6G7-E1, suggesting that 6C11-3A11 is an antibody with higher affinity.

2.2 Comparison of the Detection Sensitivities of Both Antibodies in PD-L1 Expression Analysis of Various Canine Tumors (Immunohistochemical Staining)

Using samples of canine skin squamous cell carcinoma (n=5), nasal adenocarcinoma (n=5), transitional cell carcinoma (n=5), anal sac gland carcinoma (n=5), soft tissue sarcoma (n=5) and osteosarcoma (n=5), immunohistochemical staining with rat anti-bovine PD-L1 antibody 6C11-3A11 was performed according to the method described in 2.6 of Example 1. With rat anti-bovine PD-L1 antibody 6G7-E1, immunohistochemical staining was performed in the same manner using sections derived from the same analytes. The final concentration of 6G7-E1 used on this occasion was 10 μg/ml, and biotin-labeled goat anti-rat IgM antibody (Jackson ImmunoResearch Laboratories) was used as a secondary antibody.

The results are shown in FIGS. 12 and 13. While specific signals were not observed in squamous cell carcinoma, nasal adenocarcinoma, transitional cell carcinoma, anal sac gland carcinoma and soft tissue sarcoma upon staining with 6G7-E1, satisfactory positive reactions were obtained upon staining with 6C11-3A11. On the other hand, specific signals were also obtained with 6G7-E1 in osteosarcoma but staining with 6C11-3A11 provided higher signal intensities. The PD-L1 positive rate of these tumors obtained by 6C11-3A11 staining was 100% (5 out of 5 cases) in all of the above-listed tumor species excepting soft tissue sarcoma which turned out to be PD-L1 positive at a rate of 80% (4 out of 5 cases).

Subsequently, samples of oral malignant melanoma (n=17), mammary adenocarcinoma (n=10), histiocytic sarcoma (n=10), diffuse large B-cell lymphoma (n=10) and transmissible venereal tumor (n=4) were immunohistochemically stained with 6C11-3A11 to analyze PD-L1 expression therein.

The results are shown in FIG. 14. The PD-L1 positive rate was 100% in oral malignant melanoma (17 out of 17 cases), 100% in mammary adenocarcinoma (10 out of 10 cases), 20% in histiocytic sarcoma (2 out of 10 cases), 20% in diffuse large B-cell lymphoma (2 out of 10 cases) and 0% in transmissible venereal tumor (0 out of 4 cases).

The above-described results revealed that 6C11-3A1 is superior to the existing anti-PD-L1 antibody 6G7-E1 in the detection of canine PD-L1.

Example 5

1. Introduction

Johne's disease is a bovine chronic infection caused by Mycobacterium avium subsp. paratuberculosis. In cattle affected with Johne's disease. PD-L1 expression has been confirmed in M. avium subsp. paratuberculosis-infected cells in ileal lesions which are a localized site of infection with this bacterium (Okagawa T, Konnai S, Nishimori A, Ikebuchi R, Mizorogi S. Nagata R, Kawaji S. Tanaka S, Kagawa Y. Murata S, Mori Y and Ohashi K. Infect Immun, 84:77-89, 2016). In the subject Example, immunohistochemical staining of ileai lesions of cattle with Johne's disease was performed in order to examine whether rat anti-bovine PD-L1 antibody 6C11-3A11 could be used for detecting bovine PD-L1 or not.

2. Materials and Methods

2.1. Construction of Bovine PD-L1 Expressing Cells

The nucleotide sequence of the full-length cDNA of bovine PD-L1 gene (GenBank accession number ABS10902; Ikebuchi R. Konnai S. Shirai T, Sunden Y, Murata S, Onuma M, Ohashi K Vet. Res. 2011 Sep. 26: 42:103) was determined. Based on the resultant nucleotide sequence, bovine PD-L1 membrane expressing cells were prepared. First, for preparing a bovine PD-L1 expressing plasmid, PCR was performed using a synthesized bovine PBMC-derived cDNA as a template and primers having NheI and XhoI recognition sites added on the 5′ side (boPD-L1-EGFP F and R). The PCR products were digested with NheI (Takara) and XhoI (Takara), purified with FastGene Gel/PCR Extraction Kit (NIPPON Genetics) and cloned into pEGFP-N2 vector (Clontech) that had been subjected to similar restriction enzyme treatments. The resultant expression plasmid of interest was extracted with QIAGEN Plasmid Midi kit (Qiagen) and stored at −30° C. until use in experiments. Hereinafter, the thus prepared expression plasmid is designated as pEGFP-N2-boPD-L1.

Primer (boPD-L1-EGFP F): (SEQ ID NO: 124) CTAGCTAGCACCATGAGGATATATAGTGTCTTAAC Primer (boPD-L1-EGFP R) (SEQ ID NO: 125) CAATCTCGAGTTACAGACAGAAGATGACTGC

Bovine PD-L1 membrane expressing cells were prepared by the procedures described below. First, 2.5 μg of pEGFP-N2-boPD-L1 or pEGFP-N2 (negative control) was introduced into 4×10⁶ CHO-DG44 cells using Lipofectamine LTX (Invitrogen). Forty-eight hours later, the medium was exchanged with CD DG44 medium (Life Technologies) containing G418 (Enzo Life Science) 800 μg/ml, GlutaMAX supplement (Life Technologies) 20 ml/L, and 10% Pluronic F-68 (Life Technologies) 18 ml/L; thereafter, selection was performed simultaneously with cloning by limiting dilution (bovine PD-L1 expressing cell and EGFP expressing cell). In order to confirm the expression of bovine PD-L1 in the thus prepared bovine PD-L1 expressing cell, intracellular localization of EGFP was visualized with an inverted confocal laser microscope LSM700 (ZEISS).

2.2. Binding Specificity of Rat Anti-Bovine PD-L1 Antibody 6C11-3A11 to Bovine PD-L1

It was confirmed by flow cytometry that rat anti-bovine PD-L1 antibody 6C11-3A11 specifically binds to the bovine PD-L1 expressing cell (described above). First, rat anti-bovine PD-L1 antibody 6C11-3A11 or rat IgG2a (κ) isotype control (BD Biosciences) as a negative control was reacted with the bovine PD-L1 expressing cell or the EGFP expressing cell (negative control) at room temperature for 30 min. After washing, APC-labeled anti-rat 1 g goat antibody (Southern Biotech) was reacted at room temperature for 30 min. After washing, antibodies bound to cell surfaces were detected by FACS Verse (BD Biosciences). For every washing operation and dilution of antibody. PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich) was used.

The results are shown in FIG. 15. It was revealed that rat anti-bovine PD-L1 antibody 6C11-3A11 binds specifically to the bovine PD-L1 expressing cell.

2.3. Immunohistochemical Staining Using Tissue Samples from Infected Cattle

In order to confirm that rat anti-bovine PD-L1 antibody 6C11-3A11 is applicable to PD-L1 immunohistochemical staining of bovine tissues, immunohistociemical staining was performed with formalin-fixed, paraffin-embedded bovine tissue samples. Briefly, ilium tissue blocks from cattle which naturally developed Johne's disease (#1, presenting clinical symptoms of Johne's disease such as diarrhea and severe emaciation), cattle experimentally infected with M. avium subsp. paratuberculosis (#65, clinical symptoms such as shedding of M. avium subsp. paratuberculosis and diarrhea were observed; Okagawa T. Konnai S, Nishimori A, Ikebuchi R, Mizorogi S, Nagata R, Kawaji S. Tanaka S, Kagawa Y, Murata S, Mori Y and Ohashi K. Infect Immun, 84:77-89, 2016) and uninfected control cattle (C #6) (the blocks kindly provided by Dr. Yasuyuki Mori, National Institute of Animal Health, National Agriculture and Food Research Organization) were used for staining. According to conventional methods, the stained samples were deparaffinized and then subjected to microwave treatment (5 min, twice) in citrate buffer. Subsequently, the samples were reacted with rat anti-bovine PD-L1 antibody 6C11-3A11 (400-fold dilution) for 30 min and then with Simple Stain Mouse MAX-PO (Rat) (Nichirei Bioscience) for 30 min. Finally, the samples were reacted with diarninobenzidine (DAB) for 10 min for coloring, followed by observation with an optical microscope.

The results are shown in FIG. 16. Rat anti-bovine PD-L1 antibody 6C11-3A11 detected expression of PD-L in M. avium subsp. paratuberculosis-infected cells (confirmed by Ziehl-Neelsen staining) in ileal lesions of cattle #1 that naturally developed Johne's disease and experimentally infected cattle #65 (FIG. 16a, b ). On the other hand, PD-L1 was not expressed in the ileum of uninfected cattle (C #6), so reaction of rat anti-bovine PD-L1 antibody 6C11-3A11 (non-specific reaction) was not recognized (FIG. 16a ).

As described above, it was shown that rat anti-bovine PD-L1 antibody 6C11-3A11 can be used for detecting PD-L1 in bovine tissues by immunohistochemical staining.

All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The anti-PD-L1 antibody of the present invention is applicable to diagnosis of cancers and/or infections. Further, the anti-PD-L1 antibody of the present invention is also applicable to selection of subject animals suitable for therapy with anti-PD-L1 antibodies.

SEQUENCE LISTING FREE TEXT <SEQ ID NO: 1> SEQ ID NO: 1 shows the amino acid sequence of CDR1 of the light  chain variable region (VL) of anti-PD-L1 antibody 6C11-3A11  (IgG2a). KSISKY <SEQ ID NO: 2>

<SEQ ID NO: 3> SEQ ID NO: 3 shows the amino acid sequence of CDR1 of the heavy chain variable region (VH) of anti-PD-L1 antibody 6C11-3A11  (IgG2a). GYTFTDYI <SEQ ID NO: 4> SEQ ID NO: 4 shows the amino acid sequence of CDR2 of the VH of anti-PD-L1 antibody 6C11-3A11 (IgG2a). INPDSGGN <SEQ ID NO: 5>

<SEQ ID NO: 6>

<SEQ ID NO: 7>

<SEQ ID NO: 8> SEQ ID NO: 8 shows the amino acid sequence of the light chain  (kappa chain) constant region of anti-PD-L1 antibody 6C11-3A11  (IgG2a). RADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDST YSMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC* <SEQ ID NO: 9> SEQ ID NO: 9 shows the amino acid sequence of the heavy chain  constant region (CH) of anti-PD-L1 antibody 6C11-3A11 (IgG2a). AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLT SSVTVPSSTWSSQAVTCNVAHPASSTKVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITL TPKVTCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFK CKVNSGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNG QPQENYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK* <SEQ ID NO: 10> SEQ ID NO: 10 shows the amino acid sequence (GenBank: #V01241.1)  of the light chain (kappa chain) constant region of a rat   antibody (IgG2a). ADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDSTY SMSSTLSLTKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC* <SEQ ID NO: 11> SEQ ID NO: 11 shows the amino acid sequence (GenBank: #X16129.1)  of the light chain (kappa chain) constant region of a rat   antibody (IgG2a). RADAAPTVSIFPPSTEQLATGGASVVCLMNNFYPRDISVKWKIDGTERRDGVLDSVTDQDSKDST YSMSSTLSLSKADYESHNLYTCEVVHKTSSSPVVKSFNRNEC <SEQ ID NO: 12> SEQ ID NO: 12 shows the amino acid sequence (GenBank:   #DQ402471.1) of the light chain (kappa chain) constant region of   a rat antibody (IgG2a). AAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSM SSTLSLTKVEYERHNLYTCEVVHKTSSSPVVKSFNRNEC* <SEQ ID NO: 13> SEQ ID NO: 13 shows the amino acid sequence (GenBank: #DQ402472.1)  of the CH of a rat antibody (IgG2a). APSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSGVHTFPAVLQSGLYTLTSSVT VPSSTWSSQAVTCNVAHPASSTKVDKKIVPRECNPCGCTGSEVSSVFIFPPKTKDVLTITLTPKV TCVVVDISQNDPEVRFSWFIDDVEVHTAQTHAPEKQSNSTLRSVSELPIVHRDWLNGKTFKCKVN SGAFPAPIEKSISKPEGTPRGPQVYTMAPPKEEMTQSQVSITCMVKGFYPPDIYTEWKMNGQPQE NYKNTPPTMDTDGSYFLYSKLNVKKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK* <SEQ ID NO: 14> SEQ ID NO: 14 shows the nucleotide sequence of the VL of  anti-PD-L1 antibody 6C11-3A11 (IgG2a). ATGAGGGTCCAGATTCAGTTTTGGGGGCTTCTTCTGCTCTGGACATCAGGTATACAGTGTGATGT CCAGATGACCCAGTCTCCATCTAATCTTGCTGCCTCTCCTGGAGAAAGTGTTTCCATCAATTGCA AGGCAAGTAAGAGCATTAGCAAGTATTTAGCCTGGTATCAACAGAAACCTGGGAAAGCAAATAAG CTTCTTATCTACTCTGGGTCAACTTTGCAATCTGGAACTCCATCGAGGTTCAGTGGCAGTGGATC TGGTACAGATTTCACTCTCACCATCAGAAACCTGGAGCCTGAAGATTTTGGACTCTATTACTGTC AACAGCATAATGAATACCCGCTCACGTTCGGTTCTGGGACCAAGCTGGAGATCAAA <SEQ ID NO: 15> SEQ ID NO: 15 shows the nucleotide sequence of the VH of  anti-PD-L1 antibody 6C11-3A11 (IgG2a). ATGGGATGGATCTGTATCATCTTTCTTGTGGCAATAGCTACAGGTGCCCACTCCCAGGTCAAGCT GCTGCAGTCTGGGGCTGCACTGGTGAAGCCTGGGGACTCTGTGAAGATGTCTTGCAAAGCTTCTG GTTATACATTCACTGACTACATTATACACTGGGTGAAGCAGAGTCATGGAAAAAGCCTTGAGTGG ATTGGTTATATTAATCCTGACAGTGGTGGTAATAACTACAATGAAAAGTTCAAGAGCAAGGCCAC ATTGACTGTAGACAAATCCAGCAGCACAGCCTATATGGAGTTTAGCAGATTGACATCTGAGGATT CTGCAATCTACTACTGTGCAAGAGGGATTACCATGATGGTAGTTATTAGCCACTGGAAGTTTGAC TTCTGGGGCCCAGGAACCATGGTCACCGTGTCCTCA <SEQ ID NO: 16> SEQ ID NO: 16 shows the nucleotide sequence of the light chain  (kappa chain) constant region of anti-PD-L1 antibody 6C11-3A11  (IgG2a). CGGGCTGATGCTGCACCAACTGTATCTATCTTCCCACCATCCACGGAACAGTTAGCAACTGGAGG TGCCTCAGTCGTGTGCCTCATGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTG ATGGCACTGAACGACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACG TACAGCATGAGCAGCACCCTCTCGTTGACCAAGGCTGACTATGAAAGTCATAACCTCTATACCTG TGAGGTTGTTCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAG <SEQ ID NO: 17> SEQ ID NO: 17 shows the nucleotide sequence of the CH of  anti-PD-L1 antibody 6C11-3A11 (IgG2a). GCTGAAACAACAGCCCCATCTGTCTATCCACTGGCTCCTGGAACTGCTCTCAAAAGTAACTCCAT GGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTCACCGTGACCTGGAACTCTG GAGCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGGACTCTACACTCTCACC AGCTCAGTGACTGTACCCTCCAGCACCTGGTCCAGCCAGGCCGTCACCTGCAACGTAGCCCACCC GGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCAAGGGAATGCAATCCTTGTGGATGTACAG GCTCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGACCAAAGATGTGCTCACCATCACTCTG ACTCCTAAGGTCACGTGTGTTGTGGTAGACATTAGCCAGAATGATCCCGAGGTCCGGTTCAGCTG GTTTATAGATGACGTGGAAGTCCACACAGCTCAGACTCATGCCCCGGAGAAGCAGTCCAACAGCA CTTTACGCTCAGTCAGTGAACTCCCCATCGTGCACCGGGACTGGCTCAATGGCAAGACGTTCAAA TGCAAAGTCAACAGTGGAGCATTCCCTGCCCCCATCGAGAAAAGCATCTCCAAACCCGAAGGCAC ACCACGAGGTCCACAGGTATACACCATGGCGCCTCCCAAGGAAGAGATGACCCAGAGTCAAGTCA GTATCACCTGCATGGTAAAAGGCTTCTATCCCCCAGACATTTATACGGAGTGGAAGATGAACGGG CAGCCACAGGAAAACTACAAGAACACTCCACCTACGATGGACACAGATGGGAGTTACTTCCTCTA CAGCAAGCTCAATGTAAAGAAAGAAACATGGCAGCAGGGAAACACTTTCACGTGTTCTGTGCTGC ATGAGGGCCTGCACAACCACCATACTGAGAAGAGTCTCTCCCACTCTCCTGGTAAATGA <SEQ ID NO: 18> SEQ ID NO: 18 shows the nucleotide sequence (GenBank: #V01241.1)  of the light chain (kappa chain) constant region of a rat antibody (IgG2a). GGGCTGATGCTGCACCAACTGTATCTATCTTCCCACCATCCACGGAACAGTTAGCAACTGGAGGT GCCTCAGTCGTGTGCCTCATGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTGA TGGCACTGAACGACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACGT ACAGCATGAGCAGCACCCTCTCGTTGACCAAGGCTGACTATGAAAGTCATAACCTCTATACCTGT GAGGTTGTTCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAG <SEQ ID NO: 19> SEQ ID NO: 19 shows the nucleotide sequence (GenBank: #X16129.1)  of the light chain (kappa chain) constant region of a rat   antibody (IgG2a). CGGGCTGATGCTGCACCAACTGTATCTATCTTCCCACCATCCACGGAACAGTTAGCAACTGGAGG TGCCTCAGTCGTGTGCCTCATGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTG ATGGCACTGAACGACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACG TACAGCATGAGCAGCACCCTCTCGTTGTCCAAGGCTGACTATGAAAGTCATAACCTCTATACCTG TGAGGTTGTTCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAG <SEQ ID NO: 20> SEQ ID NO: 20 shows the nucleotide sequence (GenBank: #DQ402471.1)  of the light chain (kappa chain) constant region of a rat antibody (IgG2a). GCCGCACCAACTGTATCCATCTTCCCACCATCCATGGAACAGTTAACATCTGGAGGTGCCACAGT CGTGTGCTTCGTGAACAACTTCTATCCCAGAGACATCAGTGTCAAGTGGAAGATTGATGGCAGTG AACAACGAGATGGTGTCCTGGACAGTGTTACTGATCAGGACAGCAAAGACAGCACGTACAGCATG AGCAGCACCCTCTCGTTGACCAAGGTTGAATATGAAAGGCATAACCTCTATACCTGTGAGGTTGT TCATAAGACATCATCCTCACCCGTCGTCAAGAGCTTCAACAGGAATGAGTGTTAG <SEQ ID NO: 21> SEQ ID NO: 21 shows the nucleotide sequence (GenBank: #DQ402472.1)  of the CH of a rat antibody (IgG2a). CAGCCCCCTCTGTCTATCCACTGGCTCCTGGAACTGCTCTCAAAAGTAACTCCATGGTGACCCTG GGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTCACCGTGACCTGGAACTCTGGAGCCCTGTC CAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGGACTCTACACTCTCACCAGCTCAGTGA CTGTACCCTCCAGCACCTGGTCCAGCCAGGCCGTCACCTGCAACGTAGCCCACCCGGCCAGCAGC ACCAAGGTGGACAAGAAAATTGTGCCAAGGGAATGCAATCCTTGTGGATGTACAGGCTCAGAAGT ATCATCTGTCTTCATCTTCCCCCCAAAGACCAAAGATGTGCTCACCATCACTCTGACTCCTAAGG TCACGTGTGTTGTGGTAGACATTAGCCAGAATGATCCCGAGGTCCGGTTCAGCTGGTTTATAGAT GACGTGGAAGTCCACACAGCTCAGACTCATGCCCCGGAGAAGCAGTCCAACAGCACTTTACGCTC AGTCAGTGAACTCCCCATCGTGCACCGGGACTGGCTCAATGGCAAGACGTTCAAATGCAAAGTCA ACAGTGGAGCATTCCCTGCCCCCATCGAGAAAAGCATCTCCAAACCCGAAGGCACACCACGAGGT CCACAGGTATACACCATGGCGCCTCCCAAGGAAGAGATGACCCAGAGTCAAGTCAGTATCACCTG CATGGTAAAAGGCTTCTATCCCCCAGACATTTATACGGAGTGGAAGATGAACGGGCAGCCACAGG AAAACTACAAGAACACTCCACCTACGATGGACACAGATGGGAGTTACTTCCTCTACAGCAAGCTC AATGTAAAGAAAGAAACATGGCAGCAGGGAAACACTTTCACGTGTTCTGTGCTGCATGAGGGCCT GCACAACCACCATACTGAGAAGAGTCTCTCCCACTCTCCTGGTAAATGA <SEQ ID NOS: 22 to 27> SEQ ID NOS: 22 to 27 show the nucleotide sequences of primers cPD-L1 inner F, cPD-L1 inner R, cPD-L1 5′GSP, cPD-L1 3′GSP, cPD-L1-EGFP F and cPD-L1-EGFP R in this order. <SEQ ID NO: 28> SEQ ID NO: 28 shows the amino acid sequence of the light chain (kappa chain) constant region of a human antibody. <SEQ ID NO: 29> SEQ ID NO: 29 shows the nucleotide sequence of the light chain (kappa chain) constant region of a human antibody. <SEQ ID NO: 30> SEQ ID NO: 30 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 1). <SEQ ID NO: 31> SEQ ID NO: 31 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 1). <SEQ ID NO: 32> SEQ ID NO: 32 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 2). <SEQ ID NO: 33> SEQ ID NO: 33 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 2). <SEQ ID NO: 34> SEQ ID NO: 34 shows the amino acid sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 3). <SEQ ID NO: 35>

SEQ ID NO: 35 shows the nucleotide sequence of the CH (CH1-CH3) of a human antibody (IgG4 variant 3).

<SEQ ID NO: 36>

SEQ ID NO: 36 shows the amino acid sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 37>

SEQ ID NO: 37 shows the nucleotide sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 38>

SEQ ID NO: 38 shows the amino acid sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 39>

SEQ ID NO: 39 shows the nucleotide sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 40>

SEQ ID NO: 40 shows the amino acid sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 41>

SEQ ID NO: 41 shows the nucleotide sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 42>

SEQ ID NO: 42 shows the amino acid sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 43>

SEQ ID NO: 43 shows the nucleotide sequence of the light chain (kappa chain) constant region of a mouse antibody.

<SEQ ID NO: 44>

SEQ ID NO: 44 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG1 variant 1).

<SEQ ID NO: 45>

SEQ ID NO: 45 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG1 variant 1).

<SEQ ID NO: 46>

SEQ ID NO: 46 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG1 variant 2).

<SEQ ID NO: 47>

SEQ ID NO: 47 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG1 variant 2).

<SEQ ID NO: 48>

SEQ ID NO: 48 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2a variant 1).

<SEQ ID NO: 49>

SEQ ID NO: 49 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2a variant 1).

<SEQ ID NO: 50>

SEQ ID NO: 50 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2a variant 2).

<SEQ ID NO: 51>

SEQ ID NO: 51 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2a variant 2).

<SEQ ID NO: 52>

SEQ ID NO: 52 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2b variant 1).

<SEQ ID NO: 53>

SEQ ID NO: 53 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2b variant 1).

<SEQ ID NO: 54>

SEQ ID NO: 54 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2b variant 2).

<SEQ ID NO: 55>

SEQ ID NO: 55 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2b variant 2).

<SEQ ID NO: 56>

SEQ ID NO: 56 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c variant 1).

<SEQ ID NO: 57>

SEQ ID NO: 57 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c variant 1).

<SEQ ID NO: 58>

SEQ ID NO: 58 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c variant 2).

<SEQ ID NO: 59>

SEQ ID NO: 59 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c variant 2).

<SEQ ID NO: 60>

SEQ ID NO: 60 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c % variant 3).

<SEQ ID NO: 61>

SEQ ID NO: 61 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG2c variant 3).

<SEQ ID NO: 62>

SEQ ID NO: 62 shows the amino acid sequence of the CH (CH1-CH3) of a mouse antibody (IgG3).

<SEQ ID NO: 63>

SEQ ID NO: 63 shows the nucleotide sequence of the CH (CH1-CH3) of a mouse antibody (IgG3).

<SEQ ID NO: 64>

SEQ ID NO: 64 shows the amino acid sequence of the light chain (lambda chain) constant region of a bovine antibody.

<SEQ ID NO: 65>

SEQ ID NO: 65 shows the nucleotide sequence of the light chain (lambda chain) constant region of a bovine antibody.

<SEQ ID NO: 66>

SEQ ID NO: 66 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 1).

<SEQ ID NO: 67>

SEQ ID NO: 67 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 1).

<SEQ ID NO: 68>

SEQ ID NO: 68 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 2).

<SEQ ID NO: 69>

SEQ ID NO: 69 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 2).

<SEQ ID NO: 70>

SEQ ID NO: 70 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 3).

<SEQ ID NO: 71>

SEQ ID NO: 71 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG1 variant 3).

<SEQ ID NO: 72>

SEQ ID NO: 72 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 1).

<SEQ ID NO: 73>

SEQ ID NO: 73 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 1).

<SEQ ID NO: 74>

SEQ ID NO: 74 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 2).

<SEQ ID NO: 75>

SEQ ID NO: 75 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 2).

<SEQ ID NO: 76>

SEQ ID NO: 76 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 3).

<SEQ ID NO: 77>

SEQ ID NO: 77 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG2 variant 3).

<SEQ ID NO: 78>

SEQ ID NO: 78 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 1).

<SEQ ID NO: 79>

SEQ ID NO: 79 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 1).

<SEQ ID NO: 80>

SEQ ID NO: 80 shows the amino acid sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 2).

<SEQ ID NO: 81>

SEQ ID NO: 81 shows the nucleotide sequence of the CH (CH1-CH3) of a bovine antibody (IgG3 variant 2).

<SEQ ID NO: 82>

SEQ ID NO: 82 shows the amino acid sequence of the light chain (lambda chain) constant region of a canine antibody.

<SEQ ID NO: 83>

SEQ ID NO: 83 shows the nucleotide sequence of the light chain (lambda chain) constant region of a canine antibody.

<SEQ ID NO: 84>

SEQ ID NO: 84 shows the amino acid sequence of the CH (CH1-CH3) of a canine antibody (IgG-D).

<SEQ ID NO: 85>

SEQ ID NO: 85 shows the nucleotide sequence of the CH (CH1-CH3) of a canine antibody (IgG-D).

<SEQ ID NO: 86>

SEQ ID NO: 86 shows the amino acid sequence of the light chain (kappa chain) constant region of an ovine antibody.

<SEQ ID NO: 87>

SEQ ID NO: 87 shows the nucleotide sequence of the light chain (kappa chain) constant region of an ovine antibody.

<SEQ ID NO: 88>

SEQ ID NO: 88 shows the amino acid sequence of the light chain (lambda chain) constant region of an ovine antibody.

<SEQ ID NO: 89>

SEQ ID NO: 89 shows the nucleotide sequence of the light chain (lambda chain) constant region of an ovine antibody.

<SEQ ID NO: 90>

SEQ ID NO: 90 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody (IgG1).

<SEQ ID NO: 91>

SEQ ID NO: 91 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody (IgG1).

<SEQ ID NO: 92>

SEQ ID NO: 92 shows the amino acid sequence of the CH (CH1-CH3) of an ovine antibody (IgG2).

<SEQ ID NO: 93>

SEQ ID NO: 93 shows the nucleotide sequence of the CH (CH1-CH3) of an ovine antibody (IgG2).

<SEQ ID NO: 94>

SEQ ID NO: 94 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG1^(a)).

<SEQ ID NO: 95>

SEQ ID NO: 95 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG1^(a)).

<SEQ ID NO: 96>

SEQ ID NO: 96 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG1^(b)).

<SEQ ID NO: 97>

SEQ ID NO: 97 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG1^(b)).

<SEQ ID NO: 98>

SEQ ID NO: 98 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG2^(a)).

<SEQ ID NO: 99>

SEQ ID NO: 99 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG2^(a)).

<SEQ ID NO: 100>

SEQ ID NO: 100 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG2^(b)).

<SEQ ID NO: 101>

SEQ ID NO: 101 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG2^(b)).

<SEQ ID NO: 102>

SEQ ID NO: 102 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG3).

<SEQ ID NO: 103>

SEQ ID NO: 103 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG3).

<SEQ ID NO: 104>

SEQ ID NO: 104 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG4^(a)).

<SEQ ID NO: 105>

SEQ ID NO: 105 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG4^(a)).

<SEQ ID NO: 106>

SEQ ID NO: 106 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG4^(b)).

<SEQ ID NO: 107>

SEQ ID NO: 107 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG4^(b)).

<SEQ ID NO: 108>

SEQ ID NO: 108 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG5^(a)).

<SEQ ID NO: 109>

SEQ ID NO: 109 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG5^(a)).

<SEQ ID NO: 110>

SEQ ID NO: 110 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG5^(b)).

<SEQ ID NO: 111>

SEQ ID NO: 111 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG5^(b)).

<SEQ ID NO: 112>

SEQ ID NO: 112 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG6^(a)).

<SEQ ID NO: 113>

SEQ ID NO: 113 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG6^(a)).

<SEQ ID NO: 114>

SEQ ID NO: 114 shows the amino acid sequence of the CH (CH1-CH3) of a porcine antibody (IgG6^(b)).

<SEQ ID NO: 115>

SEQ ID NO: 115 shows the nucleotide sequence of the CH (CH1-CH3) of a porcine antibody (IgG6^(b)).

<SEQ ID NO: 116>

SEQ ID NO: 116 shows the amino acid sequence of the light chain (estimated to be Ig lambda) constant region (CL) of a water buffalo antibody.

<SEQ ID NO: 117>

SEQ ID NO: 117 shows the nucleotide sequence of the light chain (estimated to be Ig lambda) constant region (CL) of a water buffalo antibody.

<SEQ ID NO: 118>

SEQ ID NO: 118 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG1).

<SEQ ID NO: 119>

SEQ ID NO: 119 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG1).

<SEQ ID NO: 120>

SEQ ID NO: 120 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG2).

<SEQ ID NO: 121>

SEQ ID NO: 121 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG2).

<SEQ ID NO: 122>

SEQ ID NO: 122 shows the amino acid sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG3).

<SEQ ID NO: 123>

SEQ ID NO: 123 shows the nucleotide sequence of the CH (CH1-CH3) of a water buffalo antibody (estimated to be IgG3).

<SEQ ID NO: 124>

SEQ ID NO: 124 shows the nucleotide sequence of primer boPD-L1-EGFP F.

<SEQ ID NO: 125>

SEQ ID NO: 125 shows the nucleotide sequence of primer boPD-L1-EGFP R. 

The invention claimed is:
 1. An anti-PD-L1 antibody comprising (a) a light chain comprising CDR1 having the amino acid sequence of KSISKY (SEQ ID NO: 1), CDR2 having the amino acid sequence of SGS and CDR3 having the amino acid sequence of QQHNEYPLT (SEQ ID NO: 2) and (b) a heavy chain comprising CDR1 having the amino acid sequence of GYTFTDYI (SEQ ID NO: 3), CDR2 having the amino acid sequence of INPDSGGN (SEQ ID NO: 4) and CDR3 having the amino acid sequence of ARGITMMVVISHWKFDF (SEQ ID NO: 5).
 2. The antibody of claim 1, which is from rat.
 3. The antibody of claim 2, which is a rat anti-bovine PD-L1 antibody.
 4. The antibody of claim 3, wherein the light chain variable region has the amino acid sequence as shown in SEQ ID NO. 6 and the heavy chain variable region has the amino acid sequence as shown in SEQ ID NO:
 7. 5. The antibody of claim 1, wherein the light chain constant region has the amino acid sequence of the constant region of kappa chain.
 6. The antibody of claim 1, wherein the heavy chain constant region has the amino acid sequence of the constant region of IgG2a.
 7. The antibody of claim 5, wherein the light chain constant region has the amino acid sequence as shown in any one of SEQ ID NOS: 8, 10 to 12 and the heavy chain constant region has the amino acid sequence as shown in SEQ ID NO: 9 or
 13. 8. The antibody of claim 1 which has a four-chain structure comprising two light chains and two heavy chains.
 9. A composition comprising the antibody of claim 1 as an active ingredient and a carrier.
 10. A method to diagnose malignant melanoma cancer comprising contacting in vitro one or more cells from a subject with the composition of claim 9; determining if said antibodies in said composition binds to said one or more cells, wherein increased binding to cancer cell as compared to a control is indicative of malignant melanoma cancer. 